Fusion polypeptide for immuno-enhancement and method for enhancing stimulation of immune response using the same

ABSTRACT

A fusion polypeptide is disclosed, which includes: (a) a mucosa targeting polypeptide; (b) a translocating peptide for translocation; and (c) a antigenic epitope. In addition, a method for enhancing a stimulation of an immune response using the aforementioned fusion polypeptide is also disclosed.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit of filing date of U.S. ProvisionalApplication Ser. No. 62/412,896, filed Oct. 26, 2016 under 35 USC §119(e)(1).

BACKGROUND 1. Field

The present disclosure relates to a fusion polypeptide, a compositioncomprising the same and a method for enhancing a stimulation of animmune response using the same.

2. Description of Related Art

Over 35 years, the antigenic peptides are critical factors in mobilizingthe immune system against foreign invaders and cancers. Most of thehighly successful treatments, including vaccines, have been madeempirically, with little immunological insight. Until 1986, it was neverconsidered that peptides could have an essential role in the control ofthe specificity of immune responses in conjunction with molecules—MHCclass I and II—best known for their involvement in transplant rejection.However, new knowledge about the chemistry and pharmacologicalproperties of antigenic peptides as well as of the molecular biology ofantigen processing, presentation, and recognition by immune cells, hasnow enabled a more rational approach to vaccine and immune enhancerdesign. Harnessing the immune system to treat disease or cancer therapywill be facilitated by a greater understanding of the origins and rolesof antigenic peptides in immunity

The roles of antigenic peptides in both the innate and adaptive immunesystems are these key players can be used for therapy against infectiousdisease, cancer and auto-immune conditions. Innate immunity is the firstline shotgun approach of defense against infections and is exemplifiedby antimicrobial peptides, also known as host defense peptides (HDPs).Adaptive immunity relies on the capacity of immune cells to distinguishbetween the body's own antigens and unwanted invaders and tumor cells.The antigen peptide belonging to the type of adaptive immunity, there isa path of the molecule from initial generation by proteolytic processingto its presentation to immune cells by major histocompatibility complex(MHC) molecules in immune system. Therefore, key players are MHC-I andMHC-II that form a noncovalent complex with antigen peptides and presentthese peptides in the context of antigen-presenting cells to T cells ofthe immune system

Although the antigenic peptides are known to be an effective manner forstimulating immune response, many researches and studies still focus onfinding novel antigenic peptides with improved effect. Therefore, anobject of the present disclosure is to find an adjuvant, which caneffectively transport to immune target organs for adaptive immunityroute to achieve the purpose of enhancing the stimulation of the immuneresponse.

SUMMARY

The present disclosure provides a fusion polypeptide, which can be usedas an enhancer for stimulating the immune response.

The present disclosure also provides a composition for enhancing astimulation of an immune response, which comprises the aforesaid fusionpolypeptide. In addition, the present disclosure further provides amethod with the aforesaid fusion polypeptide.

The fusion polypeptide of the present disclosure comprises: (a) a mucosatargeting polypeptide; (b) a first translocating peptide fortranslocation; and (c) a first antigenic epitope.

The mucosa targeting polypeptide is a binding epitope facilitating thebinding of the fusion polypeptide to a receptor in a subject in need.The first translocating peptide for translocation is used as a carrierwhich is able to facilitate cytosolic localization and antigenpresentation. The first antigenic epitope can enhance theimmune-modulating activity. When the fusion polypeptide of the presentdisclosure comprises the aforesaid three components, the fusionpolypeptide can be effectively delivered into a subject in need andenhance immune response to endogenously process target antigens.

Furthermore, another fusion polypeptide of the present disclosurecomprises: (b) a first translocating peptide for translocation; and (c)a first antigenic epitope selected from SEQ ID NOs: 5 and 6. (E622,E713).

The first translocating peptide for translocation is used as a carrierwhich is able to facilitate cytosolic localization and antigenpresentation. The first antigenic epitope is a modified antigenicepitope, which can enhance the immune-modulating activity. Especially,the sequence shown by SEQ ID NOs: 5 and 6 (i.e. E622 and E713) ismodified and different from the known polypeptides of viral proteins ofE6 and E7, which can further enhance the immune-modulating activity.When the fusion polypeptide of the present disclosure comprises theaforesaid two components, the fusion polypeptide can be effectivelydelivered into a subject in need and enhance immune response toendogenously process target antigens.

In addition, the present disclosure also provides a composition forenhancing a stimulation of an immune response, comprising: a vaccine andany one of the aforesaid fusion polypeptide. Herein, the vaccine maycomprise: a second translocation peptide for translocation and a secondantigenic epitope. The second translocation peptide is similar to thefirst translocation peptide, and the second antigenic epitope is similarto the second antigenic epitope. Thus, the descriptions about the secondtranslocation peptide and the second antigenic epitope are not repeatedagain. Furthermore, in the present disclosure, the first antigenicepitope and the second antigenic epitope has to be compatible to eachother. For example, both the first antigenic epitope and the secondantigenic epitope are HPV antigenic epitopes; but the first antigenicepitope and the second antigenic epitope is not necessarily the same.Or, both the first antigenic epitope and the second antigenic epitopeare Myostatin epitopes; but the first antigenic epitope and the secondantigenic epitope is not necessarily the same. Or, both the firstantigenic epitope and the second antigenic epitope are PRRSV antigenicepitopes; but the first antigenic epitope and the second antigenicepitope is not necessarily the same.

Moreover, the present disclosure also provides the method for enhancinga stimulation of an immune response, comprising: administering theaforesaid composition to a subject in need. Herein, the subject can bemammalian, such as human, pig, etc.

In one embodiment of the present disclosure, the antigenic epitope canbe an HPV antigenic epitope, a Myostatin epitope, or a PRRSV antigenicepitope. In another embodiment of the present disclosure, thecomposition is orally administered to the subject in need.

Other objects, advantages, and novel features of the disclosure willbecome more apparent from the following detailed description.

EMBODIMENT

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory scheme of action.

The present disclosure is related to a platform for generating someimmune-modulating chimeric polypeptides evolved in local mucosa systems,including the type-I and type-II mucosa immune system that contains amucosa targeting polypeptide which can be a M-cell or an epithelial celltargeting domain, a translocating peptide for translocation which can befrom pseudomonas exotoxin, and a antigenic epitope which can be a Th1antigenic epitope.

We survey the immune responses against the antigenic epitopes ofchimeric polypeptides. Especially, the immune responses can be elicitedwhen the fusion polypeptide of the present disclosure is administeredthrough orally administration. The fusion polypeptide of the presentdisclosure evolves the mucosal immune system in the intestine.Hereinafter, there are three groups of PE-based fusion antigens todemonstration the present disclosure that their specific immunities canbe enhanced.

1. Mucosa Targeting Polypeptide Construct

The significant advances have been made in the identification ofM-cell-specific surface markers (Kim S H et al., J Immunol. 2010 Nov.15; 185(10):5787-95). In the oral route, it should comprise the M-cellor epithelial cell ligand for mucosal targeting (Azizi et al., PLoSPathog. 2010 Nov. 11; 6(11); K J Syrjänen, J Clin Pathol. 2002 October;55(10): 721-728; Roy C. et al., GI Motility online (2006)doi:10.1038/gimo15). We have explored efficient recombinant productionof several bio-polypeptides by exploiting the natural peptides as afusion partner polypeptide.

The following embodiments use three kind peptides of binding epitopes asexamples of the M-cell target peptides in type-I mucosa immune system.Also, one kind peptide of binding epitope is as an example of theepithelial cell targeting peptide in type-II mucosa immune system (Aziziet al., PLoS Pathog. 2010 Nov. 11; 6(11); K J Syrjänen, J Clin Pathol.2002 October; 55(10): 721-728; Roy C. et al., GI Motility online (2006)doi:10.1038/gimo15; Mulder D J et al., Dig Dis Sci. 2012 March;57(3):630-42). The M-cell target peptides used in the following examplesare shown in Table 1. The epithelial cell targeting peptide is HPVL2-200, as shown in Table 2) for epithelial targeting ligand (Mulder D Jet al., Dig Dis Sci. 2012 March; 57(3):630-42).

TABLE 1 The M-cell targeting polypeptides in type-I mucosa immune system SEQ Name ID of NO. peptideAmino acid codon 01 CO-1 SFHQLPARSPAPLQ 02 DQ-2 SSFHLFHHLPARAPLAPSELQP03 RV-3 STPFHPLPARKPLPLQP

TABLE 2 The intestine epithelial cell targeting peptide SEQ Name ID ofNO. peptide Amino acid codon 04 L2-200 EFHMVD GMSIRAKRRKRASATQLYKTCfrom HPV L2 KQAGTCPPDIIPKVEGKTIAEQILQYGSM protein)GVFFGGLGIGTGSGTGGRTGYIPLGTRPP TATDTLAPVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTDT TPAILDINNNTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMD TKDEL LE *: EF, VD and LE shown initalics are the restriction enzyme sites, EcoR1 and Xho1, which areadded for subcloning purpose.

1-1. Construction of M-Cell Targeting Polypeptide

The CO-1 peptide core sequence, SFHQLPARSPAPLQ (SEQ ID NO. 01), ischosen as described in Kim S H et al., J Immunol. 2010 Nov. 15;185(10):5787-95. The DQ-2 and RV-3 amino acid code sequence are searchedby Basic Local Alignment Search Tool (BLAST) to find the regions tomimic CO-1 sequence. The program compares protein sequences to sequencedatabases and calculates the statistical significance. From the aminoacid codon sequence, we can find two CO-1-like peptides, DQ-2 (SEQ IDNO. 02): SFHLFHHLPARAPLAPL in Daedalea quercina Fr. (Polyporus-likeFugus), and RV-3 (SEQ ID NO. 03): STPFHPLPARKPLP in Dasypusnovemcinctus.

To design and generate a DNA codon sequence without DNA template, thecodon substitutions without altering the original amino acid sequence ofthe selected peptide segment was made for avoiding spurious restrictionsites and for optimal expression in E. coli. Restriction site linkerswere added at the ends of the peptide segment-encoding DNA sequence,EcoR1 and Xho1 at 5′end and 3′end, for insertion and sub-cloning intovector plasmids. The DNA fragment was generated by PCR and multipleprimers extension. The PCR DNAs, as shown in Table 3, encoded aboveamino acid sequences were respectively subcloned in to E. coli vectorplasmids by recombinant technique.

All synthesized and/or subcloned nucleotide fragments were analyzed byrestriction enzyme cutting and electrophoresis to check if they were ofthe expected sizes and right cutting sites. Once a sample had beenobtained, DNA sequences were produced automatically by machine and theresult displayed on computer. Sequence analysis performed to identifythe sequence of nucleotides in a nucleic acid, or amino acids in apolypeptide.

TABLE 3 The DNA sequence encoded M-cell target  peptide epitopes SEQName ID of NO. DNA DNA codon 05 CO-1 GAATTCAGCAGCTTTCATCTGTTCCACCATCTGCCAGCGCGTGCGCCATTAGCGCCTTCTGAATT ACAGCCCCTCGAG 06 DQ-2GAATTCAGCAGCTTTCATCTGTTCCACCATCTG CCAGCGCGTGCGCCATTAGCGCCTTCTGAATTACAGCCCCTCGAG 07 RV-3 GAATTCTCTACTCCTTTCCACCCATTGCCTGCCCGCAAACCATTGCCTCTGGTGCCCCTCGAG

1-2. Construction of Intestine Epithelial Targeting Polypeptide

L2-200 DNA fragment encoding the N-terminal domain (aa 1 to 200;“L2-200”) of HPV16 L2 protein was synthesized by multi-step PCR usingprimer pairs listed in Table 6. The primer pairs 1-10 generated DNAfragments of size 82 bp, 147 bp, 219 bp, 285 bp, 354 bp, 420 bp, 492 bp,552 bp, and 613 bp, respectively. The final DNA product was digested byrestriction enzymes Mun I and Sal I to isolate a 630 bp large DNAfragment, which was then subcloned into an EcoRI and Xho I digestedvector.

TABLE 4 Nucleotide Nucleotide No. of sequence of SEQ sequence of SEQTarget primer Fw.* the forward ID Rv.** the reverse ID peptide pairsprimer primer NO. primer primer NO. L2-200 Pair 1 F1 gttgacccggttgg 55R1 aggtcggagcaccagcgt 56 tccgtccgacccgt cgatgaaggaggtttcttcccatcgtttccctgg aaccagggaaac ttgaa Pair 2 F2 gacaccctggctcc 57 R2accggaaacgtccggcg 58 ggttcgtccgccgc ggatggacggaacggag tgaccgttgacccggtcggagcaccagc gttggt Pair 3 F3 tacatcccgctggg 59 R3 agccggggtagtgtcggt60 tacccgtccgccga ggaggtggtgatggagaa ccgctaccgacac accggaaacgtccggcctggctccg Pair 4 F4 ggtaccggttccgg 61 R4 tacggtagtaacggtgttgt 62tactggcggtcgta tgttgatgtccaggatagc ccggttacatcccg cggggtagtgtc ctgggtPair 5 F5 ggttctatgggtgttt 63 R5 cgggtcggtgaaggtcgg 64 tcttcggcggtctggttgttgtgagtggttacgg ggcatcggtaccg tagtaacggt gttccggt Pair 6 F6ggtaaaaccatcgc 65 R6 ggtttcagccggggtcgg 66 tgaacagatcctgccggttgcagaacggacgg aatacggttctatgg  gtcggtgaaggt gtgtt Pair 7 F7ggtacctgcccgcc 67 R7 ggagatggtggaagagg 68 ggacatcatcccgaacagggtgaagtgaccac aagttgaaggtaaa cggtttcagccggggt accatcgct Pair 8 F8acccagctgtacaa 69 R8 gtccatcgggatttcttcgt 70 aacctgcaaacagagttgtgggtggagatggt gctggtacctgccc ggaaga gccg Pair 9 F9 ccgtgctaaacgtc71 R9 attatttttctcgagcagttc 72 gtaaacgtgcttcc gtctttggtgtccatcgggagctacccagctgta tttcttc caaaa Pair F10 cccgaattccatatg 73 R10attatttttctcgagcagttc 74 10 gtcgacggtatgtc gtctttggtgtccatcgggacatccgtgctaaac tttcttc gtcgt *Fw: Forward. **Rv: Reversed.

TABLE 5 The DNA sequence encoded L2-200 target polypeptide SEQ Name IDof NO. DNA DNA codon 08 L2-200 GAATTCCATATGGTCGACGGTATGTCCATCCGTGCTA(from  AACGTCGTAAACGTGCTTCCGCTACCCAGCTGTACA HPV L2 AAACCTGCAAACAGGCTGGTACCTGCCCGCCGGAC protein)ATCATCCCGAAAGTTGAAGGTAAAACCATCGCTGAACAGATCCTGCAATACGGTTCTATGGGTGTTTTCTTCGGCGGTCTGGGCATCGGTACCGGTTCCGGTACTGGCGGTCGTACCGGTTACATCCCGCTGGGTACCCGTCCGCCGACCGCTACCGACACCCTGGCTCCGGTTCGTCCGCCGCTGACCGTTGACCCGGTTGGTCCGTCCGACCCGTCCATCGTTTCCCTGGTTGAAGAAACCTCCTTCATCGACGCTGGTGCTCCGACCTCCGTTCCGTCCATCCCGCCGGACGTTTCCGGTTTCTCCATCACCACCTCCACCGACACTACCCCGGCTATCCTGGACATCAACAACAACACCGTTACTACCGTAACCACTCACAACAACCCGACCTTCACCGACCCGTCCGTTCTGCAACCGCCGACCCCGGCTGAAACCGGTGGTCACTTCACCCTGTCCTCTTCCACCATCTCCACCCACAACTACGAAGAAATCCCGATGG ACACCAAAGACGAACTGCTCGAG

As shown in Tables 1 and 2, the mucosa targeting polypeptide used in thefusion polypeptide of the present disclosure may be an M-cell targetingpolypeptide (such as CO1, DQ2 and RV3) or an intestine epithelialtargeting polypeptide (such as L2-200). The polypeptides CO1, DQ2, RV3and L2-200 respectively have an amino acid sequence represented by SEQID NOs: 1 to 4.

In the present disclosure, the antigenic epitope comprised in the fusionpolypeptide of the present disclosure is a Th1 antigenic epitope.Examples of the Th1 antigenic epitope can be an HPV antigenic epitope, aMyostatin epitope, or a PRRSV antigenic epitope. Hereinafter, thesyntheses of the HPV antigenic epitope, the Myostatin epitope and thePRRSV antigenic epitope are described in detail.

2. The HPV Th1 Epitopes and its Modified Polypeptides of Viral Proteinsof E6 and E7

In the present disclosure, the HPV antigenic epitope can be an E7peptide sequence or an E6 peptide sequence of human papillomavirus type16.

Sarkar A. K. et al (2005) suggest that cellular immune responsesspecific to the E6 and E7 peptides have a role in the protectiveimmunity against HPV-associated CIN (Sarkar A K et al., Gynecol Oncol.2005 December; 99 (3 Suppl 1):S251-61). A fused peptide E601, SEQ ID NO.9: EFVDQLLRREVFCGFRDLVYDFAFSDLKLPQLCTELKLPQLCTELLE was introduced sincethe peptide Q15L (QLLRREVYDFAFRDL) and V10C (VYDFAFRDLC) of HPV-16 E6had a good CMI response.

A fused peptide E701, SEQ ID NO. 11:EFVDQAEPDOAEPDRARAHYNIRARAHYNILRAHYNIVIFRAHYNIVIF LE was synthesized,according to Tindle R. W et al in 1995 (Tindle R. W et al., Clin ExpImmunol. 1995; 101:265-271). They report that an ISCAR(=Immunostimulatory Carrier) with a BT5 peptide of HPV16 E7 whichcontains the linear B epitope 44QAEPD48, the Th epitope 48DRAHYNI54, andthe overlapping CTL epitope 49RAHYNIVTF57, has a good therapeuticeffect.

The E622 and E713 were modified from E601 and E701-polypeptide sequencesaccording to the proteasome cleavage site prediction software(http://www.imtech.res.in/raghava/pcleavage/): an SVM based method forprediction of constitutive proteasome and immunoproteasome cleavagesites in antigenic sequences for Th1 pathway. The polypeptides sequenceof E601, E622, E701 and E713 are shown as Table 6.

TABLE 6 The Th1 epitopes and its modified polypeptides of viral proteins of E6 and E7 Name SEQ of ID pep- NO. tideAmino acid codon 09 E601 EFVD QLLRREVFCGFRDLVYDFAFSDLKLPQLCTELKLPQ LCTELLE 10 E622 EFVDKDELREVYNFAFLLVLRREVYDKDELLLLLEDRQLLRREVFCGFRDLLEDRVYDFAFSDLKLPQLCTELKLPQLC TELKDELKDELVLLLLE 11 E701 EFVDQAEPDQAEPDRARAHYNIRARAHYNILRAHYNIVIF RAHYNIVIF LE 12 E713 EFVDQAEPDQAEPDRDELVLRARAHYNIRARAHYNILED RLLVLRAHYNIVIFRAHYNIVIFKDELLV LE *:EF, VD and LE shown in italics are the restriction enzyme sites, EcoR1and Xho1, which are added for subcloning purpose.

The DNA encoded of the E601, E622, E701 and E713 was designed. Codonsubstitutions without altering the original amino acid sequence of theselected peptide segment were made for avoiding spurious restrictionsites and for optimal expression in E. coli. Restriction site linkerswere added at the ends of the peptide segment-encoding DNA sequence,EcoR1, Sal1 and Xho1 at 5′end and 3′end, for insertion and sub-cloninginto a vector plasmid. The target DNA sequence of E601, E622, E701 andE713 were generated by PCR (polymerase chain reaction) and multipleprimers extension, the DNA condones are shown as Table 7. The DNAfragments of the modified nucleic acid sequence encoding target chimericpolypeptides were synthesized by PCR with multiple primers (U.S. Pat.No. 8,372,407). Non-DNA template PCR was performed. After the first runPCR, 0.01-1 μl of the DNA product were used as a DNA template for thesecond run PCR, in which the second primer pair was added together withdNTPs, reagents and Pfu polymerase. The remaining primer pairs weresequentially added in this manner at the subsequent runs of PCR untilthe target peptide-encoding DNA fragments were respectively synthesized(US

TABLE 7 The DNA codon sequences of various HPV  polypeptides SEQ Name IDof NO. DNA DNA sequence 13 E601 GAATTC GTCGAC CAACTGTTGCGTCGTGAAGTTTTCTGTGGCTTTCGTGATCTGGTCTATGACTTCGCCTTTAGTGATTTGAAGCTGCCACAATTGTGTACGGAACTGA AACTGCCTCAACTGTGTACAGAACTGAAGGATGAGCTG

14 E622 GAATTC GTCGAC AAAGATGAACTGCGTGAGGTGTATAACTTTGCGTTCCTGTTAGTGTTACGCCGTGAGGTTTATGAcAAGGACGAGTTGTTACTGCTGTTAGAAGATCGCCAACTGTTGCGTCGTGAAGTTTTCTGTGGCTTTCGTGATCTGTTAGAAGACCGCGTCTATGACTTCGCCTTTAGTGATTTGAAGCTGCCACAATTGTGTACGGAAC TGAAACTGCCTCAACTGTGTACAGAACTGAAGGATGAGCTGAAAGATGAATTAGTGCTGTTATTG

15 E701 GAATTC GTCGAC CAGGCGGAACCAGATCAAGCGGAACCTGACCGTGCCCGCGCACATTATAACATTCGCGCACGTGCACACTATAATCTGGAGGCGCATTATAACATTGTCATCTTCCGCGCACATTATAACATCGTCATTTTC

16 E713 GAATTC GTCGAC CAGGCGGAACCAGATCAAGCGGAACCTGACCGTGACGAGCTGGTGTTACGCGCCCGCGCACATTATAACATTCGCGCACGTGCACACTATAATCTGGAGGATCGTTTACTGGTCTTGCGTGCGCATTATAACATTGTCATCTTCCGCGCACATTATAACATCGTCATTTT CAAAGATGAGTTGCTGGTT

3. The Polypeptides of Myostatin Epitopes

Myostatin (also known as growth differentiation factor 8, abbreviatedGDF-8) is a myokine, a protein produced and released by myocytes thatacts on muscle cells' autocrine function to inhibit myogenesis: musclecell growth and differentiation. In humans, it is encoded by the MSTNgene. Myostatin is a secreted growth differentiation factor that is amember of the TGF beta protein family. Animals either lacking myostatinor treated with substances that block the activity of myostatin havesignificantly more muscle mass. Furthermore, individuals who havemutations in both copies of the myostatin gene have significantly moremuscle mass and are stronger than normal. Studies into myostatin showsthat myostatin may have therapeutic application in treating musclewasting diseases such as muscular dystrophy (Hamrick M K, IBMS BoneKEy.2010 January; 7(1):8-17).

3-1. M14 and M27 Construct

M14 peptide epitope was selected a partial fragment of myostatin bindingdomain; DFGLDCDEHSTESRCCRYPLTVDFEAFGWDWIIAPKRYKANYCSGECEFVFLQKYPHTHLVHQANPRGSAGPCCTPTLMSPINMLYFNGKEQIIYGKIP AMVVDRCGCS, which hasbeen patented in US20020127234. The M14 peptide core sequence,FLQKYPHTHLVHQA, was selected. Codon substitutions without altering theoriginal amino acid sequence of the selected peptide segment were madefor avoiding spurious restriction sites and for optimal expression in E.coli. Restriction site linkers were added at the ends of the peptidesegment-encoding DNA sequence, EcoR1-Sal1 and Xho1 at 5′end and 3′end,for insertion and sub-cloning into a vector plasmid. The DNA sequenceencoded the amino acid sequence of EFVDFLQKYPHTHLVHQALE was5′-GAATTCGTCGACGTGTTTTACAAAAATATCCTCATACGCACCTGG TCCATCAGGCGCTCGAC-3′.The DNA fragment was generated by PCR and multiple primers extension.Furthermore, the polypeptide with four repeated core sequence,EFVDVFLQKYPHTHLVHQALDVFLQKYPHTHLVHQALDVFLQKYPHTHLVHQALDVFLQKYPHTHLVHQALE (SEQ CO NO. 17), was generated with repeatedinsertion by recombinant technique.

M27 was modified polypeptide to present a Th1 epitope characteristicsaccording to the proteasome cleavage site prediction software(http://www.imtech.res.in/raghava/pcleavage/): an SVM based method forprediction of constitutive proteasome and immunoproteasome cleavagesites in antigenic sequences. There are two polypeptide fragments:HTHLVHQA peptide and EFLQKYPH peptide are fused and modified to createthe M27 chimeric polypeptide (no. 3). The HTHLVHQA peptide is searchedby Basic Local Alignment Search Tool (BLAST) to find regions ofsimilarity between biological sequences. The program compares proteinsequences to sequence databases and calculates the statisticalsignificance. Its amino acid codon sequence can be found in theCHLNCDRAFT_134428 protein of Chlorella variabilis (sequence ID: refXP_005847307). The amino acid codon sequence of EFLQKYPH peptide can befound in the ROOT PRIMORDIUM DEFECTIVE 1 isoform X4 of Cicer arietinum(sequence ID: ref XP_012571622.1). The two fusion peptide genes arerespectively subcloned in to E. coli vector plasmids and then fusedtogether by recombinant technique. The modified DNA fragment encoded ofLEP-HTHLVHQA-NVLLALQLLLEDREF, which added one Xho1 restriction enzymesite at 5′-end and a DNA linker or bridge encoded with NVLLALQLLLEDREFpeptide at 3′-end, is generated by primer extension and PCR runningmethod. This DNA linker encoded NVLLALQLL peptide is created by an SVMbased method for proteasome cleavage prediction. LEDR signal also isadded into fusion peptide in order to enhance Th1 immune response whenthe peptide is sorted nearby ER location. The DNA fragment encoded ofLEPHTHLVHQANVLLALQLLLEDREF peptide is generated by PCR technique. TheDNA fragment encoded of HTHLVHQA is inserted into the site of Xho1 andEcoR1 of E. coli plasmid vector. The DNA fragment encoded of EFLQKYPHVDwith two restriction enzyme sites, EcoR1 and Sal1, is generated by PCRtechnique. The DNA fragment encoded of the peptide ofLEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVD is fused the DNA fragment encodedof HTHLVHQA and the DNA fragment encoded of EFLQKYPHVD by recombinanttechnique. Furthermore, a DNA fragment with eight repeated insertion wascreated.

As shown in Table 8, a core of fused epitope: EFVFLQKYPHVEPHTHLVHQANVwas generated, and the amino acid sequence is similar to theEFVFLQKYPHTHLVHQAN of Myostatin domain in US20020127234. Finally, thepolypeptides sequence of M14 (SEQ ID NO. 17) and M27 (SEQ ID NO. 18) areshown as Table 8. The DNA coded M14 and M27 are shown as Table 9.

TABLE 8 The modified polypeptides of Myo14 and Myo27 SEQ Name ID of NO.peptide Amino acid codon 17 M14 EFVD VFLQKYPHTHLVHQALDVFLQKYPHTHLVHQALDVFLQKYPHTHLVHQALDVFLQKYPHT HLVHQA LE 18 M27 EFLLEPHTHLVHQANVLLALQLLLEDREFVFLQ KYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFV FLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDR EFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLL LEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPH VD *: VD, EF and LE shown in italics are therestriction enzyme sites, EcoR1 and Xho1, which are added for subcloningpurpose.

TABLE 9 The DNA code sequences of Myo14 and Myo27 SEQ Name ID of NO. DNADNA code sequence 19 M14 GAATTC GTCGAC GTGTTTTTACAAAAATATCCTCATACGCACCTGGTCCATCAGGCGCTCGACGTGTTTTT ACAAAAATATCCTCATACGCACCTGGTCCATCAGGCGCTCGACGTGTTTTTACAAAAATATCCTCATCGC ACCTGGTCCATCAGGCGCTCGACGTGTTTTTACAAAAATATCCTCATACGCACCTGGTCCATCAGGCG

20 M27 GAATTC CTC

CCACATACGCACTTAGTGCAT CAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCA CACGTCGAGCCACATACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCACACGT CGAGCCACATACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCACACGTCGAGC CACATACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCACACGTCGAGCCACAT ACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTT CTTGCAAAAATATCCACACGTCGAGCCACATACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCACACGTCGAGCCACATACGCACTTA GTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCCACACGTCGAGCCACATACGCACTTAGTGCATCAAGCGAACGTTTTGCTGGCACTGCAATTATTATTAGAAGATCGTGAATTTGTCTTCTTGCAAAAATATCC ACAC GTCGAC

4. The Fusion Polypeptides of PRRSV Epitope

Porcine reproductive and respiratory syndrome virus (PRRSV) causeschronic, economically devastating disease in pigs. Frequent mutations inthe viral genome result in viruses with immune escape mutants.Irrespective of regular vaccination, control of PRRSV remains achallenge to swine farmers. At present, enhancing the earlyimmunological mechanisms in PRRSV-infected pigs can improve preventiveor therapeutic purpose (0. J. Lopez et al., Clin Vaccine Immunol. 2007March; 14(3): 269-275). Recently, we try to develop an immune enhancerfor oral administration. Three immunomodulating polypeptides (GP317,GP417 and GP437) from PRRSV GP3 and GP4 epitopes are selected.

4-1. GP317, GP417 and GP437 Construct

GP317, GP417 and GP437 DNA fragment encoding the virus neutralizationdomain of PRRSV GP3 and GP4 proteins, as shown in Table 10, weresynthesized by multi-step PCR using primer pairs. Codon substitutionswithout altering the original amino acid sequence of the selectedpeptide segment were made for avoiding spurious restriction sites andfor optimal expression in E. coli. Restriction site linkers were addedat the ends of the peptide segment-encoding DNA sequence, EcoR1 and Xho1at 5′end and 3′end, for insertion and sub-cloning into a vector plasmid.The final DNA products, as shown in Table 11, were digested byrestriction enzymes EcoR1 and Xho1 to isolate DNA fragments, which werethen subcloned into an EcoRI and Xho I digested vector. Once a samplehad been obtained, DNA sequences were produced automatically by machineand the result displayed on computer. Sequence analysis performed toidentify the sequence of nucleotides in a nucleic acid, or amino acidsin a polypeptide.

TABLE 10 The immunomodulating polypeptide-enhancers SEQ Name ID of NO.peptide Amino acid codon 21 GP317 EFVSFSTGGSQNWTVERLLQAEFCSTSQAARQRLETGRNCSTGQAARQRLEPGRNLVLCLTSQAAQQRLE PGGNCQTSQAAHQRLEPGRNCRTSQAASQRLEPGRNCRTSQAAHQRLEPGRNCSTRQAAQQRLEPGRNLL CPTSQAAHQRRLEPGRNCSTSQAAYQRLEPGRNCPTSRAARQRLEPGRNLLCSTSQAALQRLEPGRNLCPT SQAAKQRLEPGRNLVVCLTSQAARQRLEPGRNCSTSQAASQRLEPGRNCPTSQAARQRLEPGRNVLLLCL TSQAAHQRLEPGRNLE 27 GP417EFGVSAAQEKISFGLLGVPTAQETTSIREVLEVSTAQENSPFMLGASATEEKTSLRLGASTTQETSFGKCLRPHGVSAAQGTTPFRGVSTTQENTSFGRVPTAQENVSFGLHGVPAAQKTNSFGGVPTAQENISFKEVSATQREIPFRCLRPHGVSTAQETPFRGVSTAQETIPFRGVSATHENISFGCLRPHGVSAAQESIPIRLGASAAQENTSFRG TPAAQEKIPLE 23 GP437EFLGVSAAQERIPIREVSADKEVSNEKKEISFGVSTAQGNISFGLGVSTAQEAIPFLALGVSTAQETIPFGLLGVSTAQGIISFGGVSTAQENISFGGVSTAQETISFGLLGVSTAQENISFGCLRTHEVSAAQEKISFGGVSEAQKISFGVSAAGVSAAQEEIPFGCLRPHGLPAAQEKTSFGG VSAAQEKTSFGGVSAAQEEFSFGCLRPHRVSAAQEKISFEVSALEVSAAQEKISFGVSAALGVSAAQEKNS FGCLRPHGVSAAQEKTSFGGVSAAQKKISFGLE*: VD, EF and LE shown in italics are the restriction enzyme sites,EcoR1 and Xho1, which are added for subcloning purpose.

TABLE 11 The DNA sequence encoded the immunomodulating polypeptide-enhancers SEQ Name ID of NO. peptide Amino acid codon 24GP317 GGAATTCGTGAGCTTTAGCACGGGTGGCAGCCAGAACTGGACGGTGGAACGTCTGCTGCAAGCCGAGT TCTGTAGTACTTCTCAGGCGGCGCGCCAGCGTCTGGAACCAGGGCGTAATTGTTCTACAGGCCAGGCCGC ACGTCAACGTTTAGAGCCAGGTCGCAATTTAGTTTTGTGTCTGACGAGCCAGGCCGCACAGCAGCGCTT GGAACCAGGCGGTAACTGTCAAACTTCTCAAGCGGCCCATCAACGCCTGGAACCAGGTCGCAACTGTCG CACTAGCCAAGCCGCCAGCCAACGTTTAGAGCCAGGCCGCAACTGTCGCACGAGTCAGGCGGCGCACC AACGTCTGGAACCAGGCCGTAATTGTAGTACGCGCCAAGCAGCCCAGCAACGCTTAGAACCAGGGCGCA ACCTGTTATGTCCAACTTCTCAGGCGGCCCATCAACGCCGCTTAGAACCAGGGCGTAATTGTAGCACGTC TCAAGCAGCATATCAACGTCTGGAACCAGGCCGCAACTGTCCAACTTCTCGTGCGGCACGCCAGCGCTTA GAACCAGGTCGTAATTTATTATGTTCTACTAGCCAAGCCGCATTACAGCGTTTAGAGCCAGGGCGTAACCT GTGTCCAACTAGCCAAGCAGCAAAACAACGCCTGGAGCCAGGTCGTAATTTAGTGGTCTGTTTAACGAG CCAAGCGGCGCGTCAACGCTTAGAACCAGGTCGCAATTGTTCTACTAGCCAAGCGGCCAGTCAACGTTT AGAACCAGGGCGCAACTGTCCAACGAGCCAAGCGGCGCGCCAACGTTTAGAGCCAGGGCGCAACGTTTT ATTGTTGTGTCTGACGAGTCAAGCCGCCCATCAACGTCTGGAACCAGGTCGCAATCTCGAG 25 GP417 GAATTCGGCGTGAGCGCGGCCCAGGAAAAGATCAGTTTCGGCCTGTTAGGTGTGCCAACGGCCCAAGAG ACTACAAGTATTCGCGAGGTTTTGGAAGTCAGTACTGCACAAGAAAACAGTCCATTTATGTTAGGCGCGA GTGCCACGGAGGAAAAAACGTCTTTGCGCCTGGGGGCAAGCACAACGCAGGAGACGAGTTTTGGCAAG TGTTTACGTCCACATGGGGTTTCTGCAGCCCAAGGGACGACTCCATTTCGCGGTGTCAGTACAACGCAAG AAAACACGAGTTTTGGTCGTGTCCCAACGGCACAAGAGAACGTGTCTTTTGGCCTGCATGGTGTTCCAG CAGCGCAAAAGACGAACAGCTTCGGTGGCGTTCCAACGGCACAAGAAAACATTAGTTTTAAGGAGGTTA GTGCCACGCAACGTGAAATCCCATTCCGTTGTTTACGCCCACACGGGGTTAGCACAGCCCAGGAGACTC CATTTCGCGGGGTGAGTACTGCCCAGGAGACGATCCCATTCCGTGGGGTTTCTGCAACGCATGAAAACAT CAGTTTTGGGTGTTTGCGTCCACATGGTGTCAGCGCCGCACAGGAATCTATTCCAATCCGTCTGGGCGCG AGCGCAGCCCAAGAGAATACCAGTTTTCGCGGGACACCAGCGGCACAGGAGAAAATCCCATTGGAACT CGAG 26 GP437GAATTCCTGGGCGTGAGCGCAGCCCAAGAGCGCA TCCCAATTCGCGAGGTGAGCGCCGACAAAGAGGTGAGTGCCGAGAAGAAAGAGATCTCTTTCGGGGTG AGCACCGCGCAGGGTAATATCAGTTTTGGTTTGGGCGTCAGCACCGCACAGGAGGCAATTCCATTCTTGG CACTGGGGGTCAGTACCGCCCAGGAAACTATTCCATTTGGCTTGCTGGGGGTTAGCACTGCACAAGGTAT CATTAGTTTCGGCGGGGTCTCTACTGCGCAGGAGAATATCAGCTTTGGCGGGGTTAGTACTGCGCAAGAG ACCATTAGTTTTGGTTTGCTGGGCGTTTCTACCGCCCAGGAGAATATTAGCTTTGGTTGTTTACGCACTCAT GAAGTTAGTGCCGCACAAGAGAAAATTAGCTTCGGCGGCGTTAGTGAAGCGCAAGAGAAGATTAGTTTCG GGGTCTCTGCAGCAGGCGTCAGCGCCGCCCAAGAGGAGATTCCATTTGGGTGTCTGCGCCCACACGGCC TGCCAGCGGCGCAGGAGAAAACCAGCTTCGGCGGCGTTAGTGCCGCCCAGGAAAAGACCTCTTTCGGTG GTGTCAGCGCAGCACAAGAAGAGTTCTCTTTTGGTTGTTTGCGCCCACATCGTGTTAGTGCCGCACAGGA AAAGATCAGCTTTGAAGTTAGCGCGCTGGAAGTCAGTGCCGCGCAAGAGAAGATTAGTTTTGGCGTTAGC GCGGCATTGGGTGTCAGCGCAGCACAAGAAAAGAACTCTTTCGGTTGTTTACGCCCACACGGTGTTAGC GCCGCGCAAGAGAAAACCAGCTTCGGGGGTGTTAGTGCCGCACAAAAAAAGATCAGCTTTGGGCTCGA G

As shown in Tables 6, 8 and 10, the antigenic epitope used in the fusionpolypeptide of the present disclosure may be E622, E713, Myo27, Myo14,GP317, GP417 or GP437, which respectively has a sequence shown by SEQ IDNOs: 10, 12, 17, 18, 21, 22 or 23.

In the present disclosure, a translocating peptide is comprised in thefusion polypeptide of the present disclosure as a carrier. Thetranslocating peptide can be from pseudomonas exotoxin. In one aspect ofthe present disclosure, the translocating peptide comprises apseudomonas exotoxin A fragment deleted of only domain III. Hereinafter,the synthesis of the translocating peptide is described in detail.

5. Pseudomonas Exotoxin (PE) Toxoid Carrier Constructs for DeliveredPeptide Antigens in the Intracellular Processing System

Pseudomonas exotoxin A (PE) polypeptide contains domains Ia, II, Ib andIII. It has a total of 613 amino acids, the full length PE-DNA fragmentwas published (Liao C W et al., Applied Microbiology and Biotechnology,July 1995, Volume 43, Issue 3, pp 498-507) and the subcloned strains arestored in Liao Lab. To investigate whether the C-terminal deletions ofthe PE polypeptide fragment would impact the immunogenicity of PE-fusedTh1 epitopes, A series of DNA fragments encoding various lengths ofPseudomonas exotoxin A polypeptide were synthesized using primer pairslisted in Table 12: PE-425, PE-407, PE-49. The term “PE-N” stands for aPE fragment consisting of as 1-N, in which N is an integer. For example,“PE-425” is a PE fragment consisting of as 1-425, and “PE-407” is a PEconsisting of as 1-407.

5-1. Example 1: pPE49 Plasmid

PE49 target gene is cloned in pET15b plasmids under control of strongbacteriophage T7 transcription and (optionally) translation signals;expression is induced by providing a source of T7 RNA polymerase in thehost cell. Plasmid pPE49 was constructed as follows. A 159 bp DNAfragment comprising a nucleotide sequence encoding PE as 1-49 wassynthesized by PCR using the primer pair PE-F1 and PE-R1, as shown inTable 12. The 165 bp PCR product was digested by Xho1 I and Nde I toisolate a 159 bp fragment. It was then subcloned into the 5.9 kb largeDNA fragment which was cut from pET15b with Xho1 I and Nde I to generateplasmid pPE-49 (6079 bp).

5-2. Example 2: pPE407 Plasmid

PE407 target gene is cloned in pET15b plasmids under control of strongbacteriophage T7 transcription and (optionally) translation signals;expression is induced by providing a source of T7 RNA polymerase in thehost cell. Plasmid pPE407 was constructed as follows. A 1224 bp DNAfragment comprising a nucleotide sequence encoding PE aa 1-407 wassynthesized by PCR using the primer pair PE-F1 and PE-R2 as shown inTable 12. The 1238 bp PCR product was digested by Xho1 I and Nde I toisolate a 1224 bp fragment. It was then subcloned into the 5.9 kb largeDNA fragment which was cut from pET15b with Xho1 I and Nde I to generateplasmid pPE-407 (7159 bp).

5-3. Example 3: pPE425 Plasmid

PE425 target gene is cloned in pET15b plasmids under control of strongbacteriophage T7 transcription and (optionally) translation signals;expression is induced by providing a source of T7 RNA polymerase in thehost cell. Plasmid pPE425 was constructed as follows. A 1299 bp DNAfragment comprising a nucleotide sequence encoding PE as 1-425 wassynthesized by PCR using the primer pair PE-F1 and PE-R3 as shown inTable 12. The 1299 bp PCR product was digested by Xho1 I and Nde I toisolate a 1293 bp fragment. It was then subcloned into the 5.9 kb largeDNA fragment which was cut from pET15b with Xho1 I and Nde I to generateplasmid pPE-425 (7213 bp).

The nucleotide and amino acid sequences of the above PE fragments arepoly-His epitopes, flanked by linkers MGSSHHHHHH and LEHHHHHHZ at 5′-and 3′- (or N- and C-) ends, respectively.

TABLE 12 Nucleotide Tar- sequence of SEQ Nucleotide se- SEQ get Fw.*the forward ID Rv.** quence of the ID PE primer primer No. primerreverse primer No. PE49 PE-F1 ccccatatggc 75 PE-R1 tttctcgagttgaat 76cgaagaagct tccatggagtagttc atcactccctggccg ttgg PE407 PE-F2 ccccatatggc77 PE-R2 tttctcgaggaattc 78 cgaagaagct gacgtcgccgccgtc gccgaggaactccgPE425 PE-F3 ccccatatggc 79 PE-R3 tttctcgaggaattc 80 cgaagaagctcgcctggagcagccg ctccaccg *Fw: Forward. **Rv: Reversed.

The target DNA fragment of PE-fused antigenic epitopes can be insertedinto Xho1 restriction enzyme site of pPE407 and pPE425.

6. Plasmid Constructions for the Mucosal Targeting Fused Polypeptides ofImmune Enhancer 6-1. Plasmid Vector Construction for Subcloning

PE49-3, plasmid construction for insertion of the DNA fragment ofM-cells target peptide epitopes. The plasmid is original from the pPE49plasmid. It was modified by PCR and recombinant manipulation. Theprimers and sequence modification were shown in Tables 13 and 14.

TABLE 13 Nucleotide Tar- Fw.* sequence of SEQ Rv.** Nucleotide se- SEQget pri- the forward ID pri- quence of the ID PE mer primer No. merreverse primer No. PE49- PE- tataccatggc 81 PE-R4 tttctcgaggaatt 82 3 F4cgaacaattgg cttccatgcagtag tggacctc tgcagcacgccc *Fw: Forward. **Rv:Reversed.

TABLE 14 The difference of N-terminal amino acid sequenceand DNA sequences and its restriction sites be- tween pPE49 and pPE49-3The N-terminal amino acid sequence of P49:MGSSHHHHHHSSGLVPRGSAMAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMEFLEHM The N-terminal amino acid sequence of P49-3:                    MAEQLVDLWNECAKACVLDLKDGVRSS RMSVDPAIADTNGQGVLHYCMEFLEHM The 5′-end pP49 DNA sequence behind T7 promotor site direction:ATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCC            Nde1GCGCGGCAGCG CCATG GCCGAAGAAGCTTTCGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCGCATGAGCGTCGACCCGGCCATCGCCGACACCAACGGCCAGGG              Nco1 EcoR1 Xho1  Nde1 CGTGCTGCACTACT CCATG GAATTC CTCGAGCATATG GCCGAAG HindIII AAGCTT TCGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGA                                  Sal1CCTCAAGGACGGCGTGCGTTCCAGCCGCATGAGC GTCGACThe 5′-end pP49-3 DNA sequence behind T7 promotor site direction:                            Nco1       Mfe1 TTTTGTTTAACTTTAAGAAGGAGATATACCATGG CCGAA CAATTG GT GGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCGCATGAGCGTCGACCCGGCCATCGCCG                                   EcoR1ACACCAACGGCCAGGGCGTGCTGCACTACTGCATG GAATTC Xho1  Nde1         HindIIICTCGAGCATATG GCCGAAG AAGCTT TCGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCGCATGAGCGTCGAGCACCACCACCACCACCACTGA

6-2. Plasmid Constructions for the M-Cell Targeting Polypeptides

Three DNA fragments (CO1, DQ2, RV3) of M-cell ligand were respectivelyligated into EcoR1 and Xho1 restriction enzymes digested pPE49-3/pETplasmids so that the DNA fragment encode the M-cell ligand was inserteddown-stream of T7 promoter. Plasmids containing inserts wererespectively transformed into E. coli and clones selected for byampicillin resistance. The partial DNA sequences of the insertionportions of pP49-3-CO1, pPE49-3-DQ2, and pPE49-3-RV3 plasmids are shownin Table 15.

TABLE 15 SEQ Plasmid ID name NO. DNA sequence pP49-3-CO1 27GTTTAACTTTAAGAAGGAGATATA CCATGGC CGAACAATTGGTGGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGA CGGCGTGCGTTCCAGCCGCATGAGCGTCGACCCGGCCATCGCCGACACCAACGGCCAGG GCGTGCTGCACTACTGCATGGAATTC

TT T

CAG

CCA

CGT

CCA

C CA

CAG CTCGAG CATATGGCCGAAG AAG CTT TCGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGC GTTCCAGCCGCATGAGCGTCGAGCACCACCACCACCACCACTGA GATCCGGCTGCTAAC pP49-3-DQ2 28 GTTTAACTTTAAGAAGGAGATATACCATGGC CGAACAATTGGTGGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGA CGGCGTGCGTTCCAGCCGCATGAGCGTCGACCCGGCCATCGCCGACACCAACGGCCAGG GCGTGCTGCACTACTGCATGGAATTC

A GC

CAT

TTC

CAT

CCA

C GT

CCA

GCG

TCT

TTA

CCC CTCGAG CATATGGCCGAAG AAGCTT TCG ACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCA GCCGCATGAGCGTCGAGCACCACCACCACC ACCACTGAGATCCGGCTGCTAAC pP49-3-RV3 29 GTTTAACTTTAAGAAGGAGATATA CCATGGCCGAACAATTGGTGGACCTCTGGAACGAATG CGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCGCATGAGCGTCGA CCCGGCCATCGCCGACACCAACGGCCAGGGCGTGCTGCACTACTGCATGGAATTC

AC T

TTC

CCA

CCT

CGC

CC A

CCT

GTG

CTCGAG CATATGGC CGAAG AAGCTT TCGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGA CGGCGTGCGTTCCAGCCGCATGAGCGTCGAGCACCACCACCACCACCACTGA GATCCGGC TGCTAAC

6-3. Plasmid Construction of the Epithelial Cell Targeting Polypeptides

L2-200 of targeting ligand DNA was ligated into EcoR1 and XhoIrestriction enzymes digested pPE49-3/pET plasmid. Plasmid containinginsert was respectively transformed into E. coli and clones selected forby ampicillin resistance. The partial DNA sequences of the insertionportion of pL2-200 plasmid are shown in Table 16.

TABLE 16 SEQ Plasmid ID name NO. DNA sequence pP49-3- 30GTTTAACTTTAAGAAGGAGATATA CCATGGC L2-200 CGAACAATTGGTGGACCTCTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGA CGGCGTGCGTTCCAGCCGCATGAGCGTCGACCCGGCCATCGCCGACACCAACGGCCAGG GCGTGCTGCACTACTGCATGGAATTCCATAT GGTCGACGGTATGTCCATCCGTGCTAAAC GTCGTAAACGTGCTTCCGCTACCCAGCTGTACAAAACCTGCAAACAGGCTGGTACC TGCCCGCCGGACATCATCCCGAAAGTTGAAGGTAAAACCATCGCTGAACAGATCCT GCAATACGGTTCTATGGGTGTTTTCTTCGGCGGTCTGGGCATCGGTACCGGTTCCGG TACTGGCGGTCGTACCGGTTACATCCCGCTGGGTACCCGTCCGCCGACCGCTACCG ACACCCTGGCTCCGGTTCGTCCGCCGCTGACCGTTGACCCGGTTGGTCCGTCCGAC CCGTCCATCGTTTCCCTGGTTGAAGAAACCTCCTTCATCGACGCTGGTGCTCCGAC CTCCGTTCCGTCCATCCCGCCGGACGTTTCCGGTTTCTCCATCACCACCTCCACCG ACACTACCCCGGCTATCCTGGACATCAACAACAACACCGTTACTACCGTAACCACTCA CAACAACCCGACCTTCACCGACCCGTCCGTTCTGCAACCGCCGACCCCGGCTGAAA CCGGTGGTCACTTCACCCTGTCCTCTTCCACCATCTCCACCCACAACTACGAAGAA ATCCCGATGGACACCAAAGACGAACTG C TCGAGCATATGGCCGAAG AAGCTT TCGACCT CTGGAACGAATGCGCCAAAGCCTGCGTGCTCGACCTCAAGGACGGCGTGCGTTCCAGCCG CATGAGCGTCGAGCACCACCACCACCACCA CTGAGATCCGGCTGCTAAC

6-4. Preparation of the Expressed Mucosa Targeting Fused Polypeptides

The chimeric polypeptides had expression in E. coli, BL21(DE3) system.Codon substitutions without altering the original amino acid sequence ofthe selected peptide segment were made for avoiding spurious restrictionsites and for optimal expression in E. coli. Restriction site linkerswere added at the ends of the peptide segment-encoding DNA sequence forinsertion into the expression vector.

7. Plasmid Constructions and Preparations of the Recombinant PE-BasedFused Antigens and Immune Enhancers 7-1. PE-Based Chimeric AntigensConstruct

Two DNA fragments (E601; SEQ ID NO. 13, and E701; SEQ ID NO. 15) of HPVTh1-antigenic epitopes were respectively ligated into EcoR1 and XhoIrestriction enzymes digested pPE425 plasmids (PE toxoid vectorderivative plasmids) so that the fusion protein was added at thePE(ΔIII) fragment C-terminal. Plasmids containing inserts wererespectively transformed into E. coli and clones selected for byampicillin resistance.

Two DNA fragment (M14; SEQ ID NO. 19, and M27; SEQ ID NO. 20) of theMyostatin epitope was ligated into EcoR1 and XhoI restriction enzymesdigested pPE407 plasmids.

Three DNA fragments (GP317; SEQ ID NO. 24, GP417; SEQ ID NO 25, andGP437; SEQ ID NO. 26) of PRRSV epitopes were respectively ligated intoEcoR1 and XhoI restriction enzymes digested pPE425 plasmids (pET and PEtoxoid vector derivative plasmids) so that the fusion protein was addedat the PE(ΔIII) fragment C-terminal. Plasmids containing inserts wererespectively transformed into E. coli and clones selected for byampicillin resistance.

TABLE 17 The examples of the PE-based recombinant chimeric antigens SEQID Name of PE-based Groups NO. chimeric antigen HPV   31 PE-E601 E601,32 PE-E701 E701 M14,  33 PE-M14 M27 34 PE-M27 GP317 35 PE-GP317 GP417 36PE-GP417 GP437 37 PE-GP437 Name SEQ of ID pep- NO. tide Amino acid codon31 PE- MGSSHHHHHHSSGLVPRGSHMAEEAFDLW E601 NECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITS DGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSE WASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLIIKPTVISHRLH FPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVD QVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVS LTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQA EF QL LRREVFCGFRDLVYDFAFSDLKLPQLCTELKLPQLCTELKDEL LE HHHHHH* 32 PE- MGSSHHHHHHSSGLVPRGSHMAEEAFDLW E701NECAKACVLDLKDGVRSSRMSVDPAIADTN GQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWS LNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNE MQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDD TWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRG WEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLAL TLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAE FLGDGGDVSFSTRGTQNWTVERLLQA EF QAEPDQAEPDRARAHYNIRARAHYNLEAHY NIVIFRAHYNIVIF LE HHHHHH* 33 PE-MGSSHHHHHHSSGLVPRGSHMAEEAFDLW M14 NECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITS DGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSE WASGKVLCLLDPIDGVYNYLAQQRCNLDDTWEGKIYRYLAGNPAKHDLDIKPTVISHRLH FPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVD QVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVS LTCPVAAGECAGPADSGDALLERNYPTGAE FLGDGGDVEFVFLQKYPHTHLVHQALDVFL QKYPHTHLVHQALDVFLQKYPHTHLVHQA LDVFLQKYPHTHLVHQALEHHHHHH* 34 PE- MGSSHHHHHHSSGLVPRGSHMAEEAFDLW M27NECAKACVLDLKDGVRSSRMSVDPAIADTN GQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWS LNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNE MQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDD TWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRG WEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLAL TLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAE FLGDGGDV EFLLEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLL ALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHL VHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYP HVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDRE FVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVE HHHHHH* 35 PE- MGSSHHHHHHSSGLVPRGSHMAEEAFDLW GP317NECAKACVLDLKDGVRSSRMSVDPAIADTN GQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWS LNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNE MQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDD TWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRG WEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLAL TLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAE FLGDGGDVSFSTRGTQNWTVERLLQA EFVSFSTGGSQNWTVERLLQAEFCSTSQAARQRL ETGRNCSTGQAARQRLEPGRNLVLCLTSQAAQQRLEPGGNCQTSQAAHQRLEPGRNCRTS QAASQRLEPGRNCRTSQAAHQRLEPGRNCSTRQAAQQRLEPGRNLLCPTSQAAHQRRLEP GRNCSTSQAAYQRLEPGRNCPTSRAARQRLEPGRNLLCSTSQAALQRLEPGRNLCPTSQA AKQRLEPGRNLVVCLTSQAARQRLEPGRNCSTSQAASQRLEPGRNCPTSQAARQRLEPGR NVLLLCLTSQAAHQRLEPGRNLE HHHHHH* 36 PE-MGSSHHHHHHSSGLVPRGSHMAEEAFDLW GP417 NECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITS DGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSE WASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLH FPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVD QVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVS LTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQA EFG VSAAQEKISFGLLGVPTAQETTSIREVLEVSTAQENSPFMLGASAFEEKTSLRLGASTTQETS FGKCLRPHGVSAAQGTTPFRGVSTTQENTSFGRVPTAQENVSFGLHGVPAAQKTNSFGGV PTAQENISFKEVSATQREIPFRCLRPHGVSTAQETPFRGVSTAQETIPFRGVSATHENISFGCL RPHGVSAAQESIPIRLGASAAQENTSFRGTPAAQEKIPLE HHHHHH* 37 PE- MGSSHHHHHHSSGLVPRGSHMAEEAFDLW GP437NECAKACVLDLKDGVRSSRMSVDPAIADTN GQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWS LNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNE MQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDD TWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRG WEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLAL TLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAE FLGDGGDVSFSTRGTQNWTVERLLQA EFLGVSAAQERIPIREVSADKEVSAEKKEISFGVST AQGNISFGLGVSTAQEAIPFLALGVSTAQETIPFGLLGVSTAQGIISFGGVSTAQENISFGGVS TAQETISFGLLGVSTAQENISFGCLRTHEVSAAQEKISFGGVSEAQKISFGVSAAGVSAAQEE IPFGCLRPHGLPAAQEKTSFGGVSAAQEKTSFGGVSAAQEEFSFGCLRPHRVSAAQEKISFE VSALEVSAAQEKISFGVSAALGVSAAQEKNSFGCLRPHGVSAAQEKTSFGGVSAAQKKISF GLE HHHHHH*

7-2. PE-Based HPV Fusion Polypeptides

To develop a PE-based HPV fusion protein as a vaccine, a reverse geneticengineering method was employed to construct a highly efficient, viralfusion protein expression vector. Through injection administration, thePE-based HPV vaccine can deliver the viral polypeptides into cellsincluding antigen presenting cells (APCs) to elicit a strong immuneresponse (Liao; Cancer research 2005). Thus, the strategy for developinga PE-based fusion polypeptide was also to fuse the C-terminus of the PEfragment with a viral protein Th1 epitope such as E601 and E701polypeptides from HPV type 16 as shown in Table 6. However, any othertypes of HPV (e.g., type 18, type 35 and any other HPV types) can alsobe used as the HPV antigenic epitope.

Four DNA fragments (E601, E622, E701, and E713) of HPV antigensTh1-epitope were respectively ligated into EcoR1 and XhoI restrictionenzymes digested pPE425 plasmids (pET23a and PE toxoid vector derivativeplasmids) so that the fusion protein was added at the PE(ΔIII) fragmentC-terminal. Plasmids containing inserts were respectively transformedinto E. coli and clones selected for by ampicillin resistance.

All synthesized and/or subcloning nucleotide fragments were analyzed byrestriction enzyme cutting and electrophoresis to check if they were ofthe expected sizes and right cutting sites. Once a sample had beenobtained, DNA sequences were produced automatically by machine and theresult displayed on computer. Sequence analysis performed to identifythe sequence of nucleotides in a nucleic acid, or amino acids in apolypeptide.

7-3. PE-Based Myostatin Epitopes Fused Polypeptides Construct

The strategy of developing a PE-based fusion protein was to fuse theC-terminus of the PE fragment with a myostatin protein binding epitope(M14) to elicit anti-Myostatin antibodies for blocking the activity ofmyostatin.

Two DNA fragments (M27, M14) of Myostatin binding epitope wererespectively ligated into EcoR1 and XhoI restriction enzymes digestedpPE407 plasmids (pET23a and PE toxoid vector derivative plasmids) sothat the fusion protein was added at the PE(ΔIII) fragment C-terminal.Plasmids containing inserts were respectively transformed into E. coliand clones selected for by ampicillin resistance. Once a sample had beenobtained, DNA sequences were produced automatically by machine and theresult displayed on computer. Sequence analysis performed to identifythe sequence of nucleotides in a nucleic acid, or amino acids in apolypeptide.

7-4. PE-Based GP3 and GP4 Fused Polypeptides Construct

The strategy of developing a PE-based fusion protein was to fuse theC-terminus of the PE fragment with virus serum neutralization epitopes(GP3 and GP4) to elicit antibodies against PRRSV infection.

The DNA fragments (GP317, GP417, and GP437) were respectively ligatedinto EcoR1 and XhoI restriction enzymes digested pPE425 plasmids (pET23aand PE toxoid vector derivative plasmids) so that the fusion protein wasadded at the PE(ΔIII) fragment C-terminal. Plasmids containing insertswere respectively transformed into E. coli and clones selected for byampicillin resistance. Once a sample had been obtained, DNA sequenceswere produced automatically by machine and the result displayed oncomputer. Sequence analysis performed to identify the sequence ofnucleotides in a nucleic acid, or amino acids in a polypeptide.

8. Plasmid Construction of the PE-Based Mucosal Targeting Fused Peptides

All the constructed plasmids, as shown in Table 18, are belonged to thefusion biogenic polypeptides, which can serve an immune enhancefunctions through the mucosal targeting and Th1 immuno-proteasomeprocessing.

The larger DNA fragment containing DQ2 epitope was cleaved frompP49-3-DQ2 plasmid with HindIII and Pst1, followed by respectivelyligating into various DNA fragments, which containing PE and Th1 epitopeplasmids, such as pPE-E713, pPE-E622, pPE-M14, pPE-M37, pPE-GP317,pPE-GP417 and pPE-GP437. Those new plasmids containing DQ2 epitope werecleaved with Mfe1 and HindIII and then exchanged other two DNA fragments(P493-CO1, and RV3). The larger DNA fragment containing HPV epithelialcell targeting epitope was cleaved from pP49-3-L2-200 plasmid withHindIII and Pst1, followed by ligating with HindIII and Pst1 restrictionenzymes digested pPE-E713 or pPE-E622 plasmid which containing PE withHPV Th1 epitope.

TABLE 18 The examples of the fusion biogenic poly-peptides for immune enhance functions Group Name of immune enhancerHPV E6 and  CO1-PE-E622, CO1-PE-E713 E7 Th1- DQ2-PE-E622, DQ2-13E-E713epiopes:  RV3-PE-E622, IW3-PE-E713 E622, E713 L2-200-PE-E662, L2-200-PE-E713 Myostatin-  CO1-PE-M27, CO1-PE-M14 like DQ2-PE-M27, DQ2-PE-M14polypeptide: RV3-PE-M27, RV3-PE-M14 M27, M14 PRRSV VN-DQ2-PE-GP417, DQ2-PE-GP437 epitopes:  DQ2-PE-GP317 GP417, GP437,  GP317Name SEQ of ID pep- NO. tide amino acid codon 38 CO1-MAEQLVDLWNECAKACVLDLKDCWRSS PE- RMSVDPAIADTNGQGVLHYCMEFSFHQL E622PARSPAPLQLEHMAEEAFDLWNECAKAC VLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLT IRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKP TVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALY LAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGT GNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFST RGTQNWTVERLLQA EFVD KDEL REVYNFAFLLVLRREVYDK DELLLLLEDR QLLR REVFCGFRDL LEDR VYDFAFSDLKLPQLCTELKLPQLCTEL KDELKDELVLLLLE HHHHHH* 39 DQ2-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSSFHL E622FHHLPARAPLAPSELQPLEHMAEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAID NALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVM AQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGN PAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAE SERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQA EFVD K DELREVYNFAFLLVLRREVYDK DELLLL LEDR QLLRREVFCGFRDL LEDR VYDFAFSDLKLPQLCTELKLPQLCTEL KDELK DELVLLL LE HHHHHH* 40 RV3-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSTPFH E622PLPARKPLPLVPLEHMAEEAFDLWNECA KACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITS DGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQ LSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPR REKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHD LDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQR LVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERF VRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGG DVSFSTRGTQNWTVERLLQA EFVD KDELREVYNFAFLLVLRREVYDK DELLLLLED R QLLRREVFCGFRDL LEDR VYDFAFSDLKLPQLCTELKLPQLCTEL KDELKDEL VLLL LE HHHHHH* 41 L2-MAEQLVDLWNECAKACVIDLKDGVRS 200- SRMSVDPAIADTNGQGVLHYCM

HM PE- VDGMSIRAKRRKRASATQLYKTCKQAGT E662 CPPDIIPKVEGKTIAEQILQYGSMGVFFGGLGIGTGSGTGGRTGYIPLGTRPPTATDTLA PVRPPLTVDPVGPSDPSIVSLVEETSFIDAGAPTSVPSIPPDVSGFSITTSTDTTPAILDINN NTVTTVTTHNNPTFTDPSVLQPPTPAETGGHFTLSSSTISTHNYEEIPMDTKDELLEH MAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGN DALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPS NIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISH AGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGK IYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGW EQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARL ALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERN YPTGAEFLGDGGDVSFSTRGTQNWTVER LLQA EFVD KDELREVYNFAFLLVLRREV YDK DELLLLLEDR QLLRREVFCGFRDL LEDRVYDFAFSDLKLPQLCTELKLPQL CTEL KDELKDELVLLL LE HHHHHH* 42 CO1-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVIEYCMEFSFHQL E713PARSPAPLQLEHMAEEAFDLWNECAKAC VLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLT IRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKP TVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALY LAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGT GNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFST RGTQNWTVERLLQA EFVD QAEPDQAEPDRDELVLRARAHYNIRARAHYNIL EDR LLVL RAHYNIVIFRAHYNIVIF KDELLV L E HHHHHH*43 DQ2- MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSSFHLE713 FHHLPARAPLAPSELQPLEHMAEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAID NALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVM AQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGN PAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAE SERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQA EFVD QAEPDQAEPDRDELVLRARAHYNIRARA HYNIL EDRLLVL RAHYNIVIFRAHYNIVI F KDELLV LEHHHHHH* 44 RV-3- MAEQLVDLWNECAKACVLDLKDGVRSS PE-RMSVDPAIADTNGQGVLHYCMEFSTPFH E713 PLPARKPLPLVPLEHMAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQ GVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGS WSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVR AHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYL AQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQAC HLPLETFRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGS GGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAG ECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQA EFVD QAEP DQAEPDRDELVLRARAHYNIRARAHY NIL EDRLLVLRAHYNIVIFRAHYNIVIF K DELLV LE HHHHHH* 45 L2- MAEQLVDLWNECAKACVLDLKDGVRSS200- RMSVDPAIADTNGQGVLHYCMEFHMVD PE- GMSIRAKRRKRASATQLYKTCKQAGTCP E713PDIIPKVEGKTIAEQILQYGSMGVFFGGL GIGTGSGTGGRTGYIPLGTRPPTATDTLAPVRPPLIVDPVGPSDPSIVSLVEETSFIDAG APTSVPSIPPDVSGFSITTSTDTTPAILDINNNTVTTVTTHNNPTFTDPSVLQPPTPAETG GHFTLSSSTISTHNYEEIPMDTKDELLEHMAEEAFDLWNECAKACVLDLKDGVRSS RMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNK PVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDE LLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVL CLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEG GSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVD QVIRNALASPGSGGDLGEMIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANA DVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVER LLQA EFVD QAEPDQAEPDRDELVLRARAHYNIRARAHYNIL EDRLLVL RAHYNIV IFRAHYNIVIF KDELLV LE HHHHHH* 46 CO1-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSFHQL M14PARSPAPLQLEHMAEEAFDLWNECAKAC VLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLT IRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHM SPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKR WSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKP TVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALY LAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGT GNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDV

VFLQKYPHTHLVHQAL D VFLQKYPHT HLVHQAL D VFLQKYPHTHLVHQAL DVFLQKYPHTHLVHQA LEHHHHHH* 47 DQ2- MAEQLVDLWNECAKACVLDLKDGVRSS PE-RMSVDPAIADTNGQGVLHYCMEFSSFHL M14 FHHLPARAPLAPSELQPLEHMAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAI ADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTR QARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARD ATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDG VYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTA HQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALA SPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPV AAGECAGPADSGDALLERNYPTGAEFLG DGGDVEFVDVFLQKYPHTHLVHQALD VFLQKYPHTHLVHQALDVFLQKYPHT HLVHQALDVFLQKYPHTHLVHQA LEHHHHHH* 48 RV-3- MAEQLVDLWNECAKACVLDLKDGVRSS PE-RMSVDPAIADTNGQGVLHYCMEFSTPFH M14 PLPARKPLPLVPLEHMAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQ GVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGS WSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVR AHESNEMPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYL AQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQAC HLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGS GGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAG ECAGPADSGDALLERNYPTGAEFLGDGG DVEFVDVFLQKYPHTHLVHQALDVFLQ KYPHTHLVHQALDVFLQKYPHTHLVH QALDVFLQKYPHTHLVHQALEHHHHH H* 49 CO1- MAEQLVDLWNECAKACVLDLKDGVRS PE-SRMSVDPAIADTNGQGVLHYCMEFSFH M27 QLPARSPAPLQLEHMAEEAFDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQ GVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGS WSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVR AHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYL AQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQAC HLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGS GGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAG ECAGPADSGDALLERNYPTGAEFLGDGG DVEFLLEPHTHLVHQANVLLALQLLLED REFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQA NVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYP HVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLL EDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVH QANVLLALQLLLEDREFVFLQKYPHVEH HHHHH* 50 DQ2-MAEQLVDLWNECAKACVLDLKDGVRS PE- SRMSVDPAIADTNGQGVLHYCMEFSSF M27HLFHHLPARAPLAPSELQPLEHMAEEA FDLWNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKL AIDNALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFI HELNAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVS VVMAQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRV LAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQL EQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALT LAAAESERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTG AEFLGDGGDVEFLLEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVH QANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQK YPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQL LLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLV HQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQ KYPHVEHHHHHH* 51 RV3-MAEQLVDLWNECAKACVLDLKDGVRS PE- SRMSVDPAIADTNGQGVLHYCMEFSTP M27FHPLPARKPLPLVPLEHMAEEA FDLWN ECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAIDNAL SITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNA GNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGNSVVMAQT QPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPA KHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGYP VQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAES ERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLGD GGDVEFLLEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLL ALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEP HTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDRE FVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVEPHTHLVHQANV LLALQLLLEDREFVFLQKYPHVEPHTHLVHQANVLLALQLLLEDREFVFLQKYPHVE HHHHHH* 52 DQ2- MAEQLVDLWNECAKACVLDLKDGVRSPE- SRMSVDPAIADTNGQGVLHYCMEFSSF GP317 HLFHHLPARAPLAPSELQPLEHMAEEAFDLWNECAKACVLDLKDGVRSSRMSVD PAIADTNGQGVLHYSMVLEGGNDALKLAIDNALSITSDGLTIRLEGGVEPNKPVRYS YTRQARGSWSLNWLVPIGHEKPSNIKVFIHELNAGNQLSHMSPIYTIEMGDELLAKL ARDATFFVRAHESNEMQPTLAISHAGVSVVMAQTQPRREKRWSEWASGKVLCLLD PLDGVYNYLAQQRCNLDDTWEGKIYRVLAGNPAKHDLDIKPTVISHRLHFPEGGSL AALTAHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVI RNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAANADVV SLTCPVAAGECAGPADSGDALLERNYPTGAEFLGDGGDVSFSTRGTQNWTVERLLQA EFVSFSTGGSQNWTVERLLQAEFCSTSQAARQRLETGRNCSTGQAARQRLEPGRNL VLCLTSQAAQQRLEPGGNCQTSQAAHQRLEPGRNCRTSQAASQRLEPGRNCRTSQA AHQRLEPGRNCSTRQAAQQRLEPGRNLLCPTSQAAHQRRLEPGRNCSTSQAAYQRL EPGRNCPTSRAARQRLEPGRNLLCSTSQAALQRLEPGRNLCPTSQAAKQRLEPGRNL VVCLTSQAARQRLEPGRNCSTSQAASQRLEPGRNCPTSQAARQRLEPGRNVLLLCLT SQAAHQRLEPGRNLEHHHHHH* 53 DQ2-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSSFHL GP417FHHLPARAPLAPSELQPLEHMAEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAID NALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVM AQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGN PAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAE SERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQAEFGVSAAQEKISFGLLGVPTAQETTSIREVLEVST AQENSPFMLGASATEEKTSLRLGASTTQETSFGKCLRPHGVSAAQGTTPFRGVSTTQE NTSFGRVPTAQENVSFGLHGVPAAQKTNSFGGVPTAQENISFKEVSATQREIPFRCLR PHGVSTAQETPFRGVSTAQETIPFRGVSATHENISFGCLRPHGVSAAQESIPIRLGASAA QENTSFRGTPAAQEKIPLE HHHHHH* 54 DQ2-MAEQLVDLWNECAKACVLDLKDGVRSS PE- RMSVDPAIADTNGQGVLHYCMEFSSFHL GP437FHHLPARAPLAPSELQPLEHMAEEAFDL WNECAKACVLDLKDGVRSSRMSVDPAIADTNGQGVLHYSMVLEGGNDALKLAID NALSITSDGLTIRLEGGVEPNKPVRYSYTRQARGSWSLNWLVPIGHEKPSNIKVFIHEL NAGNQLSHMSPIYTIEMGDELLAKLARDATFFVRAHESNEMQPTLAISHAGVSVVM AQTQPRREKRWSEWASGKVLCLLDPLDGVYNYLAQQRCNLDDTWEGKIYRVLAGN PAKHDLDIKPTVISHRLHFPEGGSLAALTAHQACHLPLETFTRHRQPRGWEQLEQCGY PVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAE SERFVRQGTGNDEAGAANADVVSLTCPVAAGECAGPADSGDALLERNYPTGAEFLG DGGDVSFSTRGTQNWTVERLLQAEFLGVSAAQERIPIREVSADKEVSAEKKEISFGVS TAQGNISFGLGVSTAQEMIPFLALGVSTAQETIPFGLLGVSTAQGIISFGGVSTAQENISF GGVSTAQETISFGLLGVSTAQENISFGCLRTHEVSAAQEKISFGGVSEAQKISFGVSAA GVSAAQEEIPFGCLRPHGLPAAQEKTSFGGVSAAQEKTSFGGVSAAQEEFSFGCLRP HRVSAAQEKISFEVSALEVSAAQEKISFGVSAALGVSAAQEKNSFGCLRPHGVSAAQ EKTSFGGVSAAQKKISFGLE HHHHHH*

Among the fusion polypeptides shown in Table 18, the mucosa targetingpolypeptide is located at an N-terminal of the fusion polypeptide, theantigenic epitope is located at a C-terminal of the fusion polypeptide,and the translocation peptide is located between the mucosa targetingpolypeptide and the antigenic epitope. However, the present disclosureis not limited thereto. In other embodiments of the present disclosure,the translocating peptide is located at an N-terminal of the fusionpolypeptide, the antigenic epitope is located at a C-terminal of thefusion polypeptide, and the mucosa targeting polypeptide is locatedbetween the translocating peptide and the antigenic epitope.

9. Fusion Polypeptide Expression and Purification

The plasmids, under the control of T7 promoter, were transformed into E.coli BL21 (DE3) for expression. After the induced expression of therecombinant polypeptides, the inclusion bodies in the lysates wererecovered; inclusion granules were harvested from the insoluble fractionby differential centrifugation (1,450×g, 10 min). The pellet was thenhomogenized in TNE buffer (10 mM Tris-HCl pH7.5, 1 mM EDTA, 50 mM NaCl)containing 100 mM PMSF and 1 mg/ml DOC, and a further 1,450×gsupernatant was collected. After three harvests, the pellets werecollected from the combined supernatants by centrifugation (27,000×g, 20min). Urea soluble contaminants were removed by re-suspending in threewashes of 0.1 M Tris-HCl, pH 8.5 containing 1 M urea followed bycentrifugation (27,000×g, 20 min). The final granule preparation wassolubilized in 20 volumes of 8 M Urea in TNE, with gentle stirring forovernight at room temperature. The proteins were then purified by S200gel filtration chromatography in denatured and reduced condition (10 mMDTT) with 6 M urea in TNE buffer. Protein elution fractions wererenatured by dialysis against TNE buffers containing from 4 M to 0 Murea in a Pellicon device (Millipore, Billerica, Mass., USA).

The clones were grown up from 2 ml of glycerol storage stocks byinoculation into 500 ml flask containing 200 ml of LB with 500 μg/mlAmpicillin. The flasks were shaken at 150 rpm and 37° C., until thecultures had an OD₆₀₀ of 1.0±0.3. Aliquots of 50 ml were inoculated ineach one of eight sterilized 3000 ml flasks containing 1250 ml LBfortified with 500 μg/ml of Ampicillin and 50 ml 10% glucose, incubatedin a 37° C. rotating incubator and shaken at 150 rpm for 2-3 hours. IPTGwas then added to a final concentration of 50 ppm, and the culture wasincubated at 37° C. with shaking for another 2 hours to complete theprotein induction. The chimeric peptides were quantified by densitometryof Coomassie Blue stained SDS-PAGE. 0.03±0.003 mg of chimericpolypeptides was used for high-dose administration, and 0.01±0.0001 mgwas used for low-dose administration in the mice animal test. For each10 liters of bacterial culture about 300-400 mg of polypeptides wasobtained, which was sufficient for 3000-9000 administrations.

10. Preparation of Microspheres of Chimeric Antigens and ImmuneEnhancers for Oral Administration The Recombinant Polypeptides ofChimeric Antigens and Immune Enhancer Preparations

The polypeptides production and sample preparation performed in a class100 laminar flow. Each recombinant polypeptide solution was equivalentto 300 mg protein content. It was transferred in 3 L volumetric flaskand then added 70-80 ml of 8M urea with powder carriers as described inTable 19.

Table 19 presents the microsphere formulations. Microsphere powders wereprepared with a co-spray drying procedure using a Model L-8 device(Ohkawara Kakohki) as described in Liao et al. (2001). The spray dryingprocess was set based on several manufacturing factors: atomizer speedat 30,000 rpm, feeding rate at 1 mL/min, hot air inlet temperature of50° C., exhaust temperature of 35° C., and cyclone pressure of ˜110 to120 mm H₂O. Microspheres were stored at 4° C. (Liao et al., 2001).

TABLE 19 The formulation of microsphere powder preparation 300 mgrecombinant polypeptide  80 ml solution in 8M urea Sterile water 1920 mlHPMC-AS  10 gram Ethyl-cellulose N20  35 gram Talc  50 gram Sodiumalginate   5 gram

To test the stability of microsphere formulation, the size and surfacestructure of microspheres and an in vitro protein-release study wereanalyzed after the formulations were stored at various temperatures forat least 3 months. The dissolution test of the enteric-coatedmicrospheres was conducted according to the pH-changing method ofUSP-XXII A (United States Pharmacopoeia, 1990). Microspheres with atotal weight of 3 g were placed in a jacketed beaker containing 500 mLof 0.02 N HCl solution (pH 1.5) at 37° C. and rotated at 100 rpm. 5 mLof 0.2 M tri-sodium phosphate buffer were added to adjust the pH to 7for further dissolution experiments, and each test was performed intriplicate. During dissolution testing, 1.5 mL of the samples were takenout at 30-min intervals, which were replaced by 1.5 mL of buffersolution to maintain the volume in the dissolution vessel. Samples werecentrifuged at 15,000 g for 5 min, and the supernatants were aspiratedand stored at −20° C.

Finally, the quantity of protein released was determined by using aCoomassie protein assay.

11. Experiment: Oral Administration of M-Cell Ligand ChimericPolypeptides Enhancing the Mice Antibody Titers Against SpecificAntigens 11-1. To Demonstrate that Immune Enhancement Effect of theFusion Biogenic Polypeptides, Vaccination Program in Mouse Tests ThroughOrally Administration of Recombinant Antigens

Male ICR mice were obtained at age 4 weeks, quarantined for 1 weekbefore the study, and maintained throughout the study on libitum withpelleted food and water. Growth curve analysis were marked at 4th weeksand weighed twice a week up to 10 weeks. Males and females wereseparated at 4 weeks and littermates of the same sex placed in the samecage in order to keep the same environment for the different treatmentduring the rest of the experiment. The animals were assigned randomly togroups, which received combinations of oral dosing fusion antigen withbio-peptide enhancers as shown in Table 20. For each treatment, at leastfour mice were analyzed. Weights were plotted on growth curves and Anovastatistical analysis using Stat View software was performed.

Used for oral immunization of PE-based vaccines and/or mucosal targetingbio-peptide enhancers were 2 different dosing schedules (3 mg low doseand 6 mg high dose of recombinant antigen formulation, i.e., equal to 10mcg and 20 mcg protein concentrations). Mice were orally administeredwith one dose/one time/per week and 4 times with this microspheresolution using a blunt-tipped feeding needle inserted into theirstomachs. The positive control group was immunized by subcutaneousinjection with 0.25 mL PE-based vaccine that included 20 mcg (H dose)protein antigens at 2-week intervals for a total of four timesimmunizations.

Two weeks after each immunization, three mice were exsanguinated andblood was collected by puncturing their retroorbital plexuses. From theresults in serum and intestinal specific antibodies titers against thefusion antigen was examined. Serum was obtained by coagulation at 4° C.for 12 h followed by centrifugation. Intestine lavage samples done atnecropsy were collected by instilling 1 mL of washing buffer (PBScontaining 100 g/mL soybean trypsin inhibitor, 50 mM EDTA, 1 mM PMSF,0.5% gelatin, and 0.05% NaN₃) into the intestine. The lavage wascollected and stored at −20° C., and lung samples after necropsy weregathered by homogenizing one-half of the lung with 0.5 mL of washingbuffer. Finally, the supernatant of a lung-homogenized sample wascollected and stored at −20° C. following centrifugation. Blood sampleswere taken and the serum assayed in an ELISA for the titer of anti-E7 oranti-E6 or anti-M14 or anti-GP3 or anti-GP4 specific antibodies usingserial ten-fold dilutions. The specific IgA or IgG or IgG1 or IgG2aantibody titer was detected after the second round of immunization. ThePRRS virus neutralization assay was examined with PE-GP417, 437 &317 byoral and injection vaccine groups.

The very high dose (VH) (100 mcg protein) and high-dose injections (H)(20 mcg protein) induced similar titers after the third roundimmunization and reached a plateau after the fourth round. The low doseof injection induced a lower titer, but was still detectable at a 1:3250dilution after the fourth round immunization.

11-2. Experimental Tables

TABLE 20 The vaccination program and examination of immune enhancementeffect of the fusion biogenic polypeptides in mouse tests Table 20-1Experiment No. 1 Vaccine groups Immune enhancer one dose/1 time/per weekL dose/1 time/per weelk and 4 times orally and 4 times orally 1. Blank 0dose — 0 dose 2. PE-E6 L dose PE-E622 L dose 3. PE-E6 L dose DQ2-PE-E622L dose 4. PE-E601 L dose — 0 dose 5. PE-E6 L dose — 0 dose 6. Positivecontrol through subcutaneous injection PE-E6* or PE-E106 (4 timesinjection with H dose) PE-E6 or PE-E106 monovalent vaccine H dose (20meg total protein/dose *: PE-E6 vaccine published in Cheng WF et al.,PLoS One. 2013 Sep 13; 8(9): e71216. Table 20-2 Experiment No. 2 Vaccinegroups Immune enhancer one dose/1 time/per week L dose/1 time/per weekand 4 times orally and 4 times orally 7. Blank 0 dose — 0 dose 8.PE-E601 2 L-doses — 0 dose 9. PE-E601 L dose L2-200-PE-E622 L dose 10.PE-E601 L dose CO1-PE-E622 L dose 11. PE-E601 L dose DQ2-PE-E622 L dose12. PE-E601 L dose RV3-PE-E6222 L dose Table 20-3 Experiment No. 3Vaccine groups Immune enhancer one dose/1 time/per week L dose/1time/per week and 4 times orally and 4 times orally 13. Blank 0 dose — 0dose 14. PE-E601 2 L-doses — — 15. PE-E601 L dose CO1-PE-E601 L dose 16.PE-E601 L dose DQ2-PE-E601 L dose 17. PE-E601 L dose RV3-PE-E601 L dose18. PE-E601 L dose L2-200-PE-E601 — Table 20-4 Experiment No. 4 Vaccinegroups Immune enhancer one dose/1 time/per week L dose/1 time/per weekand 4 times orally and 4 times orally 19. Blank — — — 20. PE-E7 L dosePE425-E713 L dose 21. PE-E7 L dose DQ2-PE425-E713 L dose 22. PE-E701 Ldose — — 23. PE-E7 L dose — — 24. Positive connol through subcutaneousinjection PE-E701 (4 times injection with H dose PE-E7 monovalentvaccine H dose (20 mcg total protein/dose Table 20-5 Experiment No. 5Vaccine groups Immune enhancer one dose/1 time/per week L dose/1time/per week and 4 times orally and 4 times orally 25. Blank — — 0 dose26. PE-E701 L dose PE-E713 L dose 27. PE-E701 L dose L2-200-PE-E713 Ldose 28. PE-E701 L dose CO1-PE-E713 L dose 29. PE-E701 L doseDQ2-PE-E713 L dose 30. PE-E701 L dose RV3-PE-E713 L dose Table 20-6Experiment No. 6 Vaccine groups Immune enhancer one dose/1 time/per weekL dose/1 time/per week and 4 times orally and 4 times orally 31. PE-M142 L-doses — — 32. PE-M14 L dose CO1-PE-M14 L dose 33. PE-M14 L doseDQ2-PE-M14 L dose 34. PE-M14 L dose RV3-PE-M14 L dose 35. PE-M14 L dosePE-M27 L dose 36. PE-M14 L dose CO1-PEM27 L dose 37. PE-M14 L doseDQ2-PE-M27 L dose 38. PE-M14 L dose RV3-PE-M27 L dose 39. Blank L dose —0 dose Table 20-7 Experiment No. 7 Vaccine groups Immune enhancer onedose/1 time/per week L dose/1 time/per week and 4 times orally and 4times orally 40. Blank — — — 41. PE-GP417 L dose CO1 PE-GP417 L dose 42.PE-GP417 L dose DQ2-PE-GP417 L dose 43. PE-GP417 L dose RV3-PE-GP417 Ldose 44. PE-GP417 2 L-doses — — 45. PE-GP437 L dose CO1-PE-GP437 L dose46. PE-GP437 L dose DQ2-PE-GP437 L dose 47. PE-GP437 L dose RV3-PE-GP437L dose 48. PE-GP437 2 L-doses — — 49. Positive control throughsubcutaneous injection PE-GP417&437 (4 times injection with H dose)PE-GP417&437 bivalent vaccine H dose (20 mcg total protein/dose Table20-8 Experiment No. 8 Vaccine groups Immune enhancer one dose/1 time/perweek L dose/1 time/per week and 4 times orally and 4 times orally 50.Blank — — — 51. PE-GP317 L dose CO1-PE-GP317 L dose 52. PE-GP317 L doseDQ2-PE-GP317 L dose 53. PE-GP317 L dose RV3-PE-GP317 L dose 54. PE-GP3172 L-doses — —

11-3. Oral Immunization Experiment in Pig Model: Oral Administration ofM-Cell Ligand Chimeric Polypeptides Enhancing the Swine Antibodies TiterAgainst Specific Antigens

In the pig model, each 24 piglets were selected respectively from sixsows source in a healthy pig farming. Three piglets from each sow wereassigned and marked with ear-number randomly to the vaccinated andcontrol groups as listed in Table 21. The oral vaccine group totallycontained nine piglets, which received orally L dose (3 mg) or H dose (6mg) suspended in a 2% acetate solution at the ages 4, 6, 7, 9, 12, 14,16, 18, 21, 24, 26 and 28-day. The intramuscular administration groupcontained another three piglets that received a VH dose (100 mcg foreach antigen) of PE-GPs trivalent with two times injection. These wereimmunized at ages 14 and 28-day by intramuscular injection with 1 mL ofvaccine that included 0.5 mL of formalin-inactive broth (300 mcg totalprotein of GP417, 437, and 317 antigens) and 0.5 mL aluminum gel. Theselast 3 piglets served as controls. After accomplishment of the oral andinjection administration program, all the experiment piglets, age29-30-day, were moved and kept in groups of ten in straw-bedded pens 3.8m×4.5 m (17 m²). Air temperature and humidity were recorded twice a daythroughout the testing period. Animals were fed the same complete feedmixture.

TABLE 21 The vaccination program and examination of immune enhancementeffect of the mucosal targeting polypeptides in piglet tests Vaccinegroups (N = 3) Immune enhancer (N = 3) L dose/3 time/per week L dose/3time/per week and 12 times orally/4 weeks and 12 times orally/4 weeksBlank 0 dose — 0 dose PE-M14 L dose DQ2-PE-M14 L dose PE-M14 L doseDQ2-PE-M27 L dose PE-M14 H dose — — PE-GP417, 437, 317 L doseDQ2-PE-GP417 L dose PE-GP417, 437, 317 L dose, additional L dose withPE-GP417 PE-GP417, 437, 317 H dose — — Positive control throughintramuscular injection (N = 3) twice at ages 14 and 28-day PE-GP417,437, 317 VH dose (300 mcg trivalent vaccine total protein/dose)

The observation and monitored for grow performance and health waspreceded by a 2-day period during which the piglets were adapted to thenew environment. The data was subjected to analysis of variance (ANOVA)to obtain the effect of post-weaning age on weight gain and linearmeasurements.

Two weeks after finishing immunization program, piglet blood wascollected by puncturing their retroorbital plexuses. From the results inserum and intestinal specific antibodies titers against the fusionantigen was examined. Serum was obtained by coagulation at 4° C. for 12h followed by centrifugation. Blood samples were taken and the serumassayed in an ELISA for the titer of anti-Myo14 or anti-GP317 oranti-GP417&437 specific antibodies using serial 2.5-fold dilutions. Thespecific IgA or IgG or IgG1 or IgG2a antibody titer was detected afterthe second round of immunization. The PRRS virus neutralization assaywas examined PE-GP417, 437 &317 oral and injection vaccine groups.

11-4. Measurement of Antibody Responses by ELISA

Polystyrene microdilution plates (Costar) were coated with antigens. Theplates were coated with 100 μl per well with this antigen at aconcentration of 2 μg/mL protein in coating buffer(carbonate-bicarbonate buffer at pH 9.6). Binding antibody titersinduced the six proteins (E7, E6, Myo14, Myo27, GP3, GP4) were analyzedusing ELISA tests. Following overnight adsorption each protein antigensat 4° C., in-directed ELISA was performed as described (Liao 2003).Horse radish peroxidase (HRP)-conjugated secondary antibodies (goatanti-Mouse IgG, IgA, IgG2, IgG1) were diluted at the optimalconcentration (×1000 fold) in blocking buffer immediately before use.Note that a positive reference sample with a high antibody titer shouldbe used to determine the titers of antibodies. This reference sample wasmade into aliquots and preserved in the lyophilized state. The serumsample was diluted 500×, 2500×, 12500×, and 62500×, respectively. Theintestinal and lung samples were diluted 10-fold times for routine work.To interpret the results accurately, ELISA (E) value was determined bythe following formula: E value=D× (S−N)/(P−N), where the OD value ofinjection vaccine sample=positive control serum or lavage, N=negativecontrol serum or lavage, S=test sample, and D=sample dilution fold.

11-5. PRRSV Serum Neutralization Test by Indirect Immunofluorescence

PRRS virus neutralization test by indirect immunofluorescence assay. Allfiltered oral fluid and serum samples (0.2-μm filter) used in the NAassay were treated with UV light (254 nm) at a distance of two inchesfrom the samples in plates for 45 min and were then heat inactivated for30 min at 56° C. Each test sample was 2-fold serially diluted (1:2 to1:128) in serum-free DMEM (100 μl per well) and incubated with an equalvolume of 100 TCID50 of one of the PRRSV strains (PRRSV TC-01) for 1 hat 37° C. After incubation, 100 μl of the supernatant was transferredinto a 96-well microtiter plate containing a confluent monolayer ofAlveolar macrophage 3D4/31 (ATCC CRL-2844; Alveolar macrophage;immortalized with SV40 large T antigen); each sample was run induplicate. After 1 h of incubation, 100 μl of DMEM containing 2% horseserum and an antibiotic-antimycotic mixture was added, and the plate wasincubated for 48 h at 37° C. in a CO₂ incubator. Cells were fixed usingan acetone/Milli-Q water (8:2) mixture for 10 min at room temperature(˜20° C.), and plates were dried completely before being immunostainedas described previously. Cells were treated with anti-PRRSV nucleocapsidprotein-specific monoclonal antibody (SDOW17) (Rural Technologies, Inc.,SD) (1:5,000) for 2 h at 37° C., followed by treatment with Alexa Fluor488 conjugated goat anti-mouse IgG(H+L) (Invitrogen, CA) secondaryantibody (1:3,000). The plate was examined under a fluorescencemicroscope after mounting with glycerol-phosphate-buffered saline (PBS)(6:4). The virus-neutralizing antibody (NA) titer was determined to bethe reciprocal dilution ratio of the sample at which >90% inhibition inthe PRRSV-induced immunofluorescence was observed.

12. Result of Experiments 12-1. The Efficacy of Fusion Polypeptides asImmune Enhancers on the PE-Based E6 Oral Vaccine

The high-risk human papillomavirus E6 (hrHPV E6) protein has been widelystudied due to its implication in the process of malignanttransformation of human cells. HPV 16 E6 oncoprotein could affect theIL-18 induced IFN-γ production in human PBMCs to elucidate the possibleimmune escape mechanisms of HPV infected cervical lesion includingcervical cancer. The E6 oncoprotein of HPV-16 and HPV-18 inhibit immuneresponse by interacting with host IRF3 and TYK2 (Li S et al., Oncogene.1999 Oct. 14; 18(42):5727-37; Masaud Shah et al., Scientific Reports 5,Article number: 13446 (2015); Cheng W F et al., PLoS One. 2013 Sep. 13;8(9):e71216; Cho Y S et al., FEBS Lett. 2001 Jul. 20; 501(2-3):139-45).In our previous research, the E6-specific immune responses generated bythe PE-E6 vaccine were weaker than the E7-specific immune responses ofthe PE-E7 through injection mice model. In this disclosure, we evaluatedthe efficacy of PE-E6 oral vaccine on immune response through oraladministration in mice model. In the experiments, we demonstrated thatthe mucosal targeting epitopes, including DQ2 and RV3 had somethingenhancement activity of E6-specific immune response from fallowing dataanalysis.

In Table 22, oral administrations of PE-E6 or PE-E601 demonstrated weakimmune response against E6 or E601 antigen according to the mice serumantibodies activity data of the group no. 4 and 5 by ELISA test.However, the serum IgA and IgG antibodies activity of PE-E6 vaccine withadditional PE-E622 or DQ2-PE-E622 immune enhancer, group no. 2 and 3,could be enhanced. From the E6-specific IgG activity in serum dilution500-fold samples, the data of the group No. 3 was significantly higherthan other oral vaccine groups without enhancer additional (p<0.05).

TABLE 22 Serum levels of IgG and IgA against HPV-16 E6 in the micegroups by ELISA test Group dilx 200 dilx 500 dilx 1250 no. En- Aver-Aver- Aver- (n = 3) Vaccine hancer age Stdev age Stdev age Stdev Serumanti-E6 IgG activity in ELISA- Test 1 Blank — 0.35 0.10 0.27 0.03 0.220.02 2 PE-E6 PE- 0.70 0.11 0.45 0.06 0.19 0.06 E622 3 PE-E6 DQ2- 0.770.08 0.59 0.08 0.25 0.08 PE- E622 4 PE-E601 — 0.39 0.05 0.22 0.05 0.180.05 5 PE-E6 — 0.55 0.14 0.34 0.10 0.22 0.10 6 Inject (H — 0.90 0.050.71 0.07 0.35 0.07 dose) Serum anti-E6 IgA activity in ELISA- Test 1Blank — 0.15 0.08 0.18 0.03 0.18 0.03 2 PE-E6 PE- 0.40 0.14 0.30 0.110.25 0.06 E622 3 PE-E6 DQ2- 0.43 0.10 0.33 0.08 0.24 0.08 PE- E622 4PE-E601 — 0.27 0.05 0.20 0.05 0.18 0.05 5 PE-E6 — 0.38 0.14 0.26 0.100.19 0.10 6 Inject (H — 0.45 0.05 0.37 0.07 0.22 0.07 dose)

In the table 23, E6 or E601-specific serum IgG1 and IgG2a responses wereassayed by ELISA, 2-3 weeks after oral administration of vaccine and/oradditional immune enhancers. From the data of the group No. 3, oraladministration of PE-E6 vaccine with additional DQ2-PE-E622 immuneenhancer could elicit a good Th1 pathway according to the high ratio ofIgG2a/IgG1. The IgG2a/IgG1 ratio was 1.4-1.5 in the group no. 3 (PE-E6oral vaccine with additional DQ2-PE-E622 enhancer). It was significantlyhigher than other groups without enhancer additional (p<0.05). TheIgG2a/IgG1 ratio of the E6-specific antibodies in group no. 2 (PE-E6oral vaccine with additional PE-E622 enhancer) was 1.13, that was notstrong significantly compared with other groups which without enhanceradditional (p>0.05).

TABLE 23 Serum activity of IgGs anti-E6 or anti-E601 and its IgG2a/IgG1ratio Group IgG2 no. IgG1 Aver- IgG2a/ (n = 3) Vaccine Enhancer AverageStdev age Stdev IgG1 Anti-E6 activity in ELISA-Test 1 Blank — 0.17 0.100.09 0.03 2 PE-E6 PE-E622 0.36 0.11 0.45 0.06 1.24 3 PE-E6 DQ2-PE- 0.420.08 0.59 0.08 1.41 E622 4 PE-E601 — 0.19 0.05 0.18 0.05 0.96 5 PE-E6 —0.38 0.14 0.24 0.10 0.63 6 Inject (H — 0.48 0.05 0.42 0.07 0.08 dose)Anti-E601 activity in ELISA-Test 1 Blank — 0.15 0.08 0.18 0.03 2 PE-E6PE-E622 0.40 0.14 0.45 0.11 1.13 3 PE-E6 DQ2-PE- 0.43 0.10 0.65 0.081.51 E622 4 PE-E601 — 0.29 0.05 0.27 0.05 0.94 5 PE-E6 — 0.32 0.14 0.240.10 0.75 6 Inject (H — 0.33 0.05 0.22 0.07 0.67 dose)

In Table 24, five groups of mice were oral administration of PE-E601vaccine additional with various enhancers including PE-E601,L2-L200-PE-E622, CO1-PE-E622, DQ2-PE-E622 and RV3-PE-E622, respectively.

The immune responses of those groups of mice against E601 antigen wereexamined according to mice serum IgG1, IgG2a antibodies activity byELISA test. Through the IgG2a/IgG1 ratio data, the Th1 or Th2 immunepathway could be prospected. The IgG2a/IgG1 ratio was 1.56 or 1.46 inthe group no. 11 or 12 (PE-E601 oral vaccine with additional DQ2-PE-E622or RV3-PE-E622 enhancer), but those of the group 8 or 9 or 10 (PE-E601oral vaccine additional with PE-E601 or L2-200-PE-E622 or CO1-PE-E622enhancer) was lower than 1.05. According to the high ratio ofE601-specific IgGs and IgG2a/IgG1 data in mice experiments, theDQ2-PE-E622 and RV3-PE-E622 enhancers could strongly elicit a good Th1immunity when PE-based E601 oral vaccine administration with additionalthe enhancers. The Th1 immunity enhancing efficacy of DQ2-PE-E622 andRV3-PE-E622 were significantly different from other groups (P<0.05).

TABLE 24 Group Anti-E601 activity IgG2 no. in ELISA-Test IgG1 Aver-IgG2a/ (n = 3) Vaccine Enhancer Average Stdev age Stdev IgG1 7 Blank —0.15 0.08 0.18 0.03 8 PE-E601 2 L-doses 0.33 0.04 0.28 0.07 0.83 9PE-E601 L2-200- 0.40 0.03 0.38 0.03 0.94 PE-E622 10 PE-E601 CO1-PE- 0.420.03 0.44 0.03 1.04 E622 11 PE-E601 DQ2-PE- 0.29 0.04 0.45 0.05 1.56E622 12 PE-E601 RV3-PE- 0.31 0.03 0.45 0.04 1.46 E6222

In Table 25, five groups of mice are oral administration of PE-E601vaccine additional with various enhancers including PE-E601,L2-200-PE-E601, CO1-PE-E601, DQ2-PE-E601 and RV3-PE-E601, respectively.

The immune response of those groups of mice against E601 antigenexamined according to mice serum IgG1, IgG2a antibodies activity byELISA test. Through the IgG2a/IgG1 ratio data, the Th1 or Th2 pathwaycould be prospected. The IgG2a/IgG1 ratio is 1.24 or 1.28 in the groupno. 16 or 17 (PE-E601 oral vaccine additional with DQ2-PE-E622 orRV3-PE-E622 enhancer), but those of the group 14 or 15 or 18 (PE-E601oral vaccine additional with PE-E601 or CO1-PE-E622 or L2-200-PE-E622)was lower than 1.05. According to the high ratio of IgG2a/IgG1 data inmice experiments, the DQ2-PE-E622 and RV3-PE-E622 enhancers can elicit agood Th1 pathway when PE-based E016 oral vaccine administrationadditional with the enhancers (p<0.05).

TABLE 25 Group Anti-E601 activity IgG2 no. in ELISA-Test IgG1 Aver-IgG2a/ (n = 3) Vaccine Enhancer Average Stdev age Stdev IgG1 13 Blank —0.15 0.08 0.18 0.03 14 PE-E601 2 L-doses 0.33 0.05 0.28 0.07 0.83 15PE-E601 CO1-PE- 0.34 0.05 0.34 0.03 1.00 E601 16 PE-E601 DQ2-PE- 0.350.05 0.44 0.03 1.24 E601 17 PE-E601 RV3-PE- 0.35 0.04 0.45 0.05 1.28E601 18 PE-E601 L2-200- 0.33 0.05 0.31 0.04 0.94 PE-E601

12-2. The Effect of Fusion Biogenic Polypeptides Enhancers on thePE-Based E7 Oral Vaccine

Our previous studies indicated that a based E7 fusion protein vaccineenhanced MHC class I and II presentation of E7, leading to dramaticincreases in the number of E7-specific CD8+ and CD4+ T-cell precursorsand markedly raised titers of E7-specific antibodies. These resultsindicated that retrograde-fusion protein via the delivery domains ofexotoxins with an antigen greatly enhances in vivo antigen-specificimmunologic responses and represents a novel strategy to improve cancerinjection vaccine potency (Ebrahimpoor S et al., Iran J Allergy AsthmaImmunol. 2013 Aug. 28; 12(4):361-7.). In the present disclosure, we tryto evaluate the efficacy of PE-E7 oral vaccine on immune response ofmice test through oral administration. We performed a study on the oraladministration experiments of PE-E7 in mice immunization test that wegot similar results that were found in PE-E6 study. Orallyadministrations of PE-E7 or PE-E701 demonstrated weak immune responseagainst E7 or E701 antigen. In the present disclosure, we demonstratedthat the mucosal targeting epitopes, including DQ2 and RV3 had somethingenhancement activity of E6-specific antibodies from fallowing dataanalysis.

In Table 26, oral administrations of PE-E7 or PE-E701 demonstrated weakimmune response against E7 or E701 antigen according to the mice serumantibodies activity data of the group no. 22 and 23 by ELISA test.

TABLE 26 Group dlix 200 dilx 500 dilx 1250 no. Aver- Aver- Aver- (n = 3)Vaccine Enhancer age Stdev age Stdev age Stdev Serum anti-E7 IgGactivity in ELISA-Test 19 Blank — 0.30 0.10 0.24 0.03 0.18 0.04 20 PE-E7PE-E713 0.72 0.15 0.43 0.10 0.30 0.07 21 PE-E7 DQ2-PE- 0.93 0.05 0.550.06 0.39 0.05 E713 22 PE- — 0.44 0.07 0.22 0.06 0.19 0.05 E701 23 PE-E7— 0.68 0.14 0.34 0.10 0.22 0.10 24 Inject — 1.50 0.15 0.75 0.07 0.480.07 (H dose) Serum anti-E7 IgA activity in ELISA-Test 19 Blank — 0.210.08 0.18 0.03 0.18 0.03 20 PE-E7 PE-E713 0.50 0.16 0.30 0.10 0.25 0.0621 PE-E7 DQ2-PE- 0.60 0.12 0.33 0.05 0.24 0.08 E713 22 PE- — 0.21 0.150.20 0.07 0.18 0.05 E701 23 PE-E7 — 0.43 0.20 0.26 0.07 0.19 0.10 24Inject — 0.60 0.15 0.37 0.07 0.22 0.07 (H dose)

However, the serum IgA and IgG antibodies activity of PE-E7 vaccinegroup with additional PE-E622 or DQ2-PE-E622, group no. 20 and 21, couldbe enhanced. From the E7-specific IgG activity in serum dilution200-fold samples, the data of the group No. 21 was significantly higherthan other oral vaccine groups without enhancer additional (p<0.05).

In Table 27, five groups of mice were oral administration of PE-E701vaccine additional with various enhancers including PE-E713,L2-L200-PE-E713, CO1-PE-E713, DQ2-PE-E713 and RV3-PE-E713, respectively.

TABLE 27 Serum anti-E701 Group IgGs activity IgG2 no. in ELISA-Test IgG1Aver- IgG2a/ (n = 3) Vaccine Enhancer Average Stdev age Stdev IgG1 25Blank — 0.18 0.09 0.19 0.05 26 PE-E701 PE-E713 0.36 0.06 0.37 0.10 1.0327 PE-E701 L2-200- 0.42 0.06 0.65 0.05 1.55 PE-E713 28 PE-E701 CO1-PE-0.47 0.06 0.63 0.07 1.34 E713 29 PE-E701 DQ2-PE- 0.47 0.08 0.67 0.091.43 E713 30 PE-E701 RV3-PE- 0.48 0.06 0.69 0.05 1.44 E713

The immune responses of those groups of mice against E701 antigen wereexamined according to mice serum IgG1, IgG2a antibodies activity byELISA test. Through the IgG2a/IgG1 ratios data, the Th1 or Th2 immunepathway could be prospected. The IgG2a/IgG1 ratios were 1.55, 1.34, 1.43and 1.44 in the group no. 27, no. 28, no. 29 and no. 30 which werePE-E701 oral vaccine with additional L2-200-PE-713, CO1-PE-E713,DQ2-PE-E713, RV3-PE-E713 enhancers, respectively. The IgG2a/IgG1 ratiosof group 26 (PE-E701 oral vaccine additional with PE-E713 enhancer) waslower than 1.05. According to the high activity of E701-specific IgG2aand high IgG2a/IgG1 ratios data, the L2-200-PE-713, CO1-PE-E713,DQ2-PE-E622 and RV3-PE-E622 enhancers could strongly elicit a good Th1immunity when PE-based E713 oral vaccine administration with additionalthese enhancers in mice immunization model. The immunity enhancingefficacy of E701-specific IgG2a showed that L2-200-PE-713, CO1-PE-E713,DQ2-PE-E713 and RV3-PE-E713 groups had significantly different fromPE-E713 enhancer groups (P<0.05).

12-3. The Efficacy of Fusion Polypeptides as Immune Enhancers on thePE-Based Myostatin Oral Vaccine

Recombinant myostatin can induce immune responses to myostatin by oralroute, resulting in increasing body weight in mice. It is an importantstep towards transforming cells into edible vaccine to improve meatproduction in farm animals and combat muscle-waste genetic diseases inhuman (Zhang T et al., BMC Biotechnol. 2012 Dec. 19; 12:97; Aravind S etal., J Virol Methods. 2012 November; 185(2):234-8).

We have established a PE-based myostatin fused oral vaccine for animaluse. There were several fusion biogenic polypeptide enhancers weredeveloped for PE-based myostatin fused oral vaccine formulation. In thepresent disclosure, we demonstrated that the mucosal targeting epitopes,including CO1, DQ2 and RV3 had something enhancement activity ofmyostatin epitope-specific antibodies from fallowing data analysis.

In Tables 28 and 29, the M14-specific serum IgG and IgA activities ofELISA in serial serum dilutions (1:200-1:1250) were examined in variousserum samples.

TABLE 28 Serum anti-M14 Group IgG activity dilx 200 dilx 500 dilx 1250no. in ELISA-Test Aver- Aver- Aver- (n = 3) Vaccine Enhancer age Stdevage Stdev age Stdev 31 PE-M14 2 L-doses 0.56 0.20 0.51 0.07 0.34 0.04 32PE-M14 CO1-PE- 0.80 0.23 0.78 0.16 0.41 0.04 M14 33 PE-M14 DQ2-PE- 0.810.16 0.66 0.06 0.44 0.04 M14 34 PE-M14 RV3-PE- 0.95 0.24 0.74 0.17 0.360.06 M14 39 Blank — 0.30 0.10 0.27 0.03 0.22 0.02

TABLE 29 Serum anti-M14 Group IgA activity dilx 200 dilx 500 dilx 1250no. in ELISA-Test Aver- Aver- Aver- (n = 3) Vaccine Enhancer age Stdevage Stdev age Stdev 35 PE-M14 PE-M27 0.58 0.24 0.45 0.01 0.29 0.09 36PE-M14 CO1-PE- 0.75 0.12 0.69 0.16 0.35 0.05 M27 37 PE-M14 DQ2-PE- 0.750.06 0.64 0.07 0.33 0.03 M27 38 PE-M14 RV3-PE- 0.79 0.13 0.66 0.16 0.290.05 M27 39 Blank — 0.27 0.02 0.25 0.03 0.23 0.03

According to serial dilutions ELISA data of the group no. 31 (PE-M14vaccine group), it demonstrated that oral administration of PE-M14vaccine elicited a good M14-specific IgG and IgA serum titers in the1:200 to 1:1250 fold serum dilutions. Furthermore, the serum IgA and IgGantibodies activities of the groups of PE-M14 vaccine with additionalCO1-PE-M14, DQ2-PE-M14 and RV3-PE-M14, CO1-PE-M27, DQ2-PE-M27 andRV3-PE-M27, corresponded to the group no. 32, 33 34, 36, 37 and 38 couldbe extremely enhanced. From the M14-specific IgG activity in serumdilution 500-fold samples, the data of the group No. 32, No. 33 and No.34 were significantly higher than other oral vaccine groups (p<0.05).

In Table 30, the data demonstrated that PE-based M14 fused oral vaccinescould be enhanced by the mucosal targeting ligands, CO1, DQ2 and RV3.The serum specific-M14 antibodies activities (with serum 500dilutions-ELISA) of IgG and IgA of the groups no. 32, 33 and 34, whichtreated with additional CO1-PE-M14, DQ2-PE-M14 and RV3-PE-M14 enhancerrespectively, were slightly higher than that of activities of the groupNo. 31 (p>0.1). Furthermore, the activities of groups no. 36, 37, 38presented very high level than that of groups no. 31 or no. 35, withoutMucosal targeting ligands additional. Specifically, the IgG and IgAlevels of group no. 37 and 38 were significantly higher (p<0.05) whencompared with the control group (such as group no. 31 or 35).

TABLE 30 Value of OD405 Value of OD405 Serum anti-M14 by ELISA by ELISAGroup IgA activity (the level of (the level of no. in ELIS A-Test IgGantibody) IgA antibody) (n = 3) Vaccine Enhancer Average Stdev AverageStdev 31 PE-M14 2 L-doses 0.48 0.07 0.40 0.05 32 PE-M14 CO1-PE- 0.620.10 0.53 0.10 M14 33 PE-M14 DQ2-PE- 0.60 0.16 0.55 0.08 M14 34 PE-M14RV3-PE- 0.65 0.13 0.57 0.05 M14 35 PE-M14 PE-M27 0.51 0.07 0.45 0.08 36PE-M14 CO1-PE- 0.78 0.22 0.69 0.16 M27 37 PE-M14 DQ2-PE- 0.68 0.05 0.640.07 M27 38 PE-M14 RV3-PE- 0.75 0.10 0.66 0.06 M27 39 Blank — 0.30 0.100.27 0.1

12-4. The Efficacy of Fusion Polypeptides as Immune Enhancers on thePE-Based PRRS GP3-GP4 Epitopes Oral Vaccine

Porcine reproductive and respiratory syndrome (PRRS) causes devastatingeconomic losses due to late-term reproductive failure and severepneumonia in neonatal pigs. PRRS disease is a high-consequence animaldisease with current vaccines providing limited protection frominfection due to the high degree of genetic variation of field PRRSvirus. Serum neutralizing antibodies (NAs) considered being an importantcorrelate of protective immunity against PRRSV. The role that NA have inprotection against infection with PRRSV had been demonstrated by Lopezet al (2007). His results identified certain threshold of serum virusneutralization (SVN) titer (≥1:8) at which the dissemination of PRRSV inthe serum of a young pig would be blocked, as well as a higher threshold(≥1:32) that could imply complete protection of the animal from PRRSVinfection. We had been developed a PE-based PRRSV subunit vaccine.However, the subunit vaccine has to be improved about SVN titereliciting against PRRS disease control. In the present disclosure, thefallowing experimental result could show that the efficacy of fusionpolypeptides as oral immune enhancers could elicit SVN.

In Table 31, the data demonstrated that PE-based GPs fused oral vaccinescould be enhanced by the mucosal targeting ligands, CO1, DQ2 and RV3.

According to serial dilutions ELISA data of the group no. 44 and 48(PE-GP417 and PE-GP437 vaccine groups), it showed that oraladministration of PE-GP417 and PE-GP437 elicited a good GP417 andGP437-specific IgG serum titers in the 1:200 to 1:1250 fold serumdilutions. The serum IgG antibodies activities of PE-GP417 or PE-GP437vaccine with additional CO1-PE-GP417 or CO1-PE-GP437, DQ2-PE-GP417 orDQ2-PE-GP437 and RV3-PE-GP417 or RV3-PE-GP437 immune enhancers,corresponded to the group no. 41 or 45, 42 or 46 and 43 or 47, couldincrease the antibodies activity. From the GP417-specific IgG activityin serum dilution 500-fold samples, the data of the group No. 41, No. 42and No. 43 were significantly higher than other oral vaccine groups(p<0.02). From the GP-437-specific IgG activity in that dilution foldsamples of the group No. 46 and 47, it showed good immune enhancingeffect, but No. 45 group was not. From the result, the mucosal targetingepitopes including CO1, DQ2 and RV3 had good enhancement activity inGP417 epitope-specific antibodies, and DQ2 and RV3 had good enhancementactivity in the GP437 antibodies.

TABLE 31 Serum anti-GP417 Group & 437 IgG activity no. in ELISA-Testdilx 200 dilx 500 dilx 1250 dilx 3125 (n = 3) Vaccine Enhancer AverageStdev Average Stdev Average Stdev Average Stdev 40 Blank — 0.31 0.100.23 0.08 0.22 0.05 0.27 0.08 41 PE-GP417 CO1-PE- 0.60 0.11 0.49 0.060.28 0.06 0.22 0.06 GP417 42 PE- DQ2-PE- 0.77 0.08 0.59 0.08 0.39 0.080.22 0.03 GP417 GP417 43 PE- RV3-PE- 0.69 0.11 0.52 0.05 0.38 0.05 0.250.05 GP417 GP417 44 PE-GP417 0.52 0.13 0.34 0.10 0.25 0.11 0.28 0.05 2L-doses 45 PE-GP437 CO1-PE- 0.64 0.11 0.51 0.11 0.26 0.08 0.24 0.05GP437 46 PE- DQ2-PE- 0.85 0.12 0.63 0.08 0.44 0.09 0.32 0.03 GP437 GP43747 PE- RV3-PE- 0.75 0.10 0.54 0.10 0.38 0.10 0.28 0.05 GP437 GP437 48PE-GP437 0.58 0.14 0.34 0.10 0.22 0.10 0.25 0.06 2 L-doses 49 Inject —1.50 0.20 0.10 0.30 0.58 0.20 0.25 0.06 (H dose)

TABLE 32 Serum anti-GP417 & 437 IgA Group activity in no. ELISA-Testdilx 200 dilx 500 dilx 1250 dilx 3125 (n = 3) Vaccine Enhancer AverageStdev Average Stdev Average Stdev Average Stdev 40 Blank — 0.28 0.100.27 0.03 0.21 0.08 0.21 0.03 41 PE- CO1-PE- 0.65 0.12 0.38 0.08 0.290.05 0.24 0.04 GP417 GP417 42 PE- DQ2-PE- 0.70 0.10 0.45 0.05 0.28 0.050.26 0.10 GP417 GP417 43 PE- RV3-PE- 0.60 0.12 0.38 0.06 0.27 0.06 0.280.10 GP417 GP417 44 PE-GP417 0.50 0.11 0.31 0.13 0.25 0.10 0.22 0.10 2L-dose 45 PE- CO1-PE- 0.59 0.15 0.39 0.09 0.28 0.03 0.22 0.06 GP437GP437 46 PE- DQ2-PE- 0.81 0.11 0.45 0.10 0.31 0.05 0.27 0.07 GP437 GP43747 PE- RV3-PE- 0.72 0.14 0.48 0.11 0.34 0.05 0.27 0.05 GP437 GP437 48PE-GP437 0.53 0.10 0.29 0.13 0.26 0.05 0.22 0.09 2 L-doses 49 Inject —0.80 0.20 0.50 0.15 0.30 0.08 0.23 0.04 (H dose)

From the results shown in Table 32, the GP417 and GP437-specific IgAactivity in PE-GP417 and PE-GP437 vaccine were slightly enhanced by themucosal targeting epitopes, including CO1, DQ2 and RV3 (p>0.02).

In Tables 33 and 34, the data showed that serum virus-neutralization(VN) antibodies has increased when PE-based GP417&437 and GP317 fusedoral vaccines additional with the mucosal targeting ligands, CO1, DQ2and RV3.

TABLE 33 Serum titers of the oral vaccine immunization in the experimentgroup in mice model IgG-HASA PRRSV TC- titers 01 in (S/P) coatingAlveolar Group antigen macrophage no. Group treatment GP417 & 3D4/31 (n= 3) Oral vaccine Enhancer 437 VN titers 40 Blank — 0 0 41 PE-GP417CO1-PE-GP417 149 16 42 PE-GP417 DQ2-PE-GP417 207 32 43 PE-GP417RV3-PE-GP417 167 16 44 PE-GP417 2 L-doses 63 8 45 PE-GP437 CO1-PE-GP437161 16 46 PE-GP437 DQ2-PE-GP437 230 32 47 PE-GP437 RV3-PE-GP437 178 1648 PE-GP437 2 L-doses 64 8 49 Inject (H dose) — 500 64

TABLE 34 Serum anti-GP317 Group IgG activity no. in ELISA-Test dilx 200dilx 500 (n = 3) Vaccine Enhancer Average Stdev Average Stdev 50 Blank —0.28 0.10 0.22 0.08 51 PE-GP317 CO1-PE- 0.50 0.10 0.35 0.06 G-P317 52PE-GP317 DQ2-PE- 0.62 0.10 0.51 0.07 GP317 53 PE-GP317 RV3-PE- 0.58 0.100.44 0.07 GP317 54 PE-GP317 2 L-doses 0.52 0.10 0.46 0.11 55 Inject (H —0.88 0.12 0.68 0.10 dose) PRRSV Group Serum anti-GP317 IgG activity inIgG-ELISA TC-01 in no. ELISA-Test titers Alveolar (n = 3) Oral vaccineEnhancer (S/P) GP317 VN titers 50 Blank — 0 0 51 PE-GP317 CO1-PE-GP317141 8 52 PE-GP317 DQ2-PE-GP317 315 32 53 PE-GP317 RV3-PE-GP317 239 8 54PE-GP317 2 L-doses 261 8 55 Inject (H dose) — 500 32

From the results shown in Tables 31 and 32, only the group of No. 42,46, 52 had the highest level of VN titers. The oral administration ofPE-GP317, PE-GP414 and PE-GP437 vaccine without the immune enhancer,such as the group no. 44, 48 and 54, all showed a lower VN titers andonly 8 score. However, the DQ2 fused polypeptide, which proposed a goodmucosal targeting ligand, demonstrated a good immune enhancing efficacywhen it added into vaccine groups such as the group No. 42, 46 and 52.

13. Pigs Experiment: Oral Administration of M-Cell Ligand ChimericPolypeptides Enhancing the Swine Antibody Titer Against Specific Antigen

The vaccination program and examination of immune enhancement effect ofthe fusion biogenic polypeptides in swine tests is shown in Table 21.

In pig oral vaccine experiment of PE-M14 H-dose group without enhancer,there were no interaction effects of age x dietary program on growthperformance from weaning to 10 weeks of age. The PE-M14 H-dose group didnot affect growth performance from weaning to 10 weeks of age. It had noeffect (P>0.05) on pig weight at 10 weeks of age. However, preweaningADG (0.172 kg/day for ≤0.104 kg birth weight to 0.27 kg/day for ≥1.99 kgbirth weight), weaning weight (5.26 kg to 8.85 kg), weaning BCS (2.69 to2.93), and preweaning mortality (24.2% to 4.6%) were improved for pigsof heavier birth weight categories.

Over the entire 5-wk postweaning phase, PE-M14 H-dose oral vaccineadditional with DQ2-PE-M14 or DQ2-PE-M27 enhancer group, the piglets hada 23-24% higher weight gain (P<0.05) and showed more play behavior(4.0±0.3 vs. 2.8±0.3 freq/h, P<0.05) than that of placebo group.

TABLE 35 IgG-ELISA titers (S/P) PRRSV coating TC-01 in Vaccine group (N= 3) antigens: Alveolar L dose/3 times/per Immune enhancer (N = 3)GP417, macrophage week and 12 times L dose/3 times/per week GP437 and3D4/31 orally/4 weeks and 12 times orally/4 weeks GP 317 VN titers Blank0 dose — 0 dose 1 0 PE-GP417, L dose DQ2-PE-GP417 L dose 320 32 437, 317PE-GP417, 437, 317 L dose 160 16 and PE-GP-417 L dose PE-GP417, H dose —— 160 8 437, 317 Positive control through intramuscular injection (N =3) 640 128

From the data shown in Table 33, the IgG-ELISA and VN titers in oralvaccine groups were lower than that of injection groups. VN titers couldincrease the score when the oral vaccine additional with DQ2-PE-GP417.It showed that DQ2 fused polypeptide, which proposed a good mucosaltargeting ligand, demonstrated a good immune enhancer for PE-basedPRRSV-NT oral vaccine.

In conclusion, when the vaccine is used with a fusion polypeptide of thepresent disclosure, good immune response can be achieved when thecomposition is orally administered. Especially, when the fusionpolypeptide of the present disclosure comprises a mucosa targetingpolypeptide, the immune response is significantly increased.

Although the present disclosure has been explained in relation to itsembodiment, it is to be understood that many other possiblemodifications and variations can be made without departing from thespirit and scope of the disclosure as hereinafter claimed.

What is claimed is:
 1. A fusion polypeptide, comprising: (a) a mucosatargeting polypeptide; (b) a first translocating peptide fortranslocation; and (c) a first antigenic epitope.
 2. The fusionpolypeptide of claim 1, wherein the mucosa targeting polypeptide islocated at an N-terminal of the fusion polypeptide, the first antigenicepitope is located at a C-terminal of the fusion polypeptide, and thefirst translocation peptide is located between the mucosa targetingpolypeptide and the first antigenic epitope.
 3. The fusion polypeptideof claim 1, wherein the mucosa targeting polypeptide is an M-celltargeting polypeptide or an intestine epithelial targeting polypeptide.4. The fusion polypeptide of claim 1, wherein the mucosa targetingpolypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1to
 4. 5. The fusion polypeptide of claim 1, wherein the firsttranslocating peptide is from pseudomonas exotoxin.
 6. The fusionpolypeptide of claim 1, wherein the first translocating peptidecomprises a pseudomonas exotoxin A fragment deleted of only domain III.7. The fusion polypeptide of claim 1, wherein the first antigenicepitope is a Th1 antigenic epitope.
 8. The fusion polypeptide of claim1, wherein the first antigenic epitope is an HPV antigenic epitope, aMyostatin epitope, or a PRRSV antigenic epitope.
 9. The fusionpolypeptide of claim 8, wherein the HPV antigenic epitope is an E7peptide sequence or an E6 peptide sequence of human papillomavirus type16.
 10. The fusion polypeptide of claim 1, wherein the first antigenicepitope is selected from SEQ ID NOs: 10, 12, 17, 18, 21, 22 and
 23. 11.A method for enhancing a stimulation of an immune response, comprising:administering a composition to a subject in need, wherein thecomposition comprises a vaccine and a fusion polypeptide, and the fusionpolypeptide comprises: (a) a mucosa targeting polypeptide; (b) a firsttranslocating peptide for translocation; and (c) a first antigenicepitope.
 12. The method of claim 11, wherein the mucosa targetingpolypeptide is located at an N-terminal of the fusion polypeptide, thefirst antigenic epitope is located at a C-terminal of the fusionpolypeptide, and the first translocation peptide is located between themucosa targeting polypeptide and the first antigenic epitope.
 13. Themethod of claim 11, wherein the mucosa targeting polypeptide is a M-celltargeting polypeptide or an intestine epithelial targeting polypeptide.14. The method of claim 11, wherein the mucosa targeting polypeptidecomprises an amino acid sequence selected from SEQ ID NOs: 1 to
 4. 15.The method of claim 11, wherein the first translocating peptide is frompseudomonas exotoxin.
 16. The method of claim 11, wherein the firsttranslocating peptide comprises a pseudomonas exotoxin A fragmentdeleted of only domain III.
 17. The method of claim 11, wherein thefirst antigenic epitope is a Th1 antigenic epitope.
 18. The method ofclaim 11, wherein the first antigenic epitope is an HPV antigenicepitope, a Myostatin epitope, or a PRRSV antigenic epitope.
 19. Themethod of claim 18, wherein the HPV antigenic epitope is an E7 peptidesequence or an E6 peptide sequence of human papillomavirus type
 16. 20.The method of claim 11, wherein the first antigenic epitope is selectedfrom SEQ ID NOs: 10, 12, 17, 18, 21, 22 and
 23. 21. The method of claim11, wherein the composition is orally administered to the subject inneed.
 22. The method of claim 11, wherein the vaccine comprises: asecond translocation peptide for translocation and a second antigenicepitope.
 23. The method of claim 22, wherein the second translocatingpeptide is from pseudomonas exotoxin.
 24. The method of claim 22,wherein the second translocating peptide comprises a pseudomonasexotoxin A fragment deleted of only domain III.
 25. The method of claim22, wherein the second antigenic epitope is a Th1 antigenic epitope. 26.The method of claim 22, wherein the second antigenic epitope is an HPVantigenic epitope, a Myostatin epitope, or a PRRSV antigenic epitope.27. The method of claim 11, wherein the vaccine is an anti-HPV vaccine,an anti-Myostatin vaccine or an anti-PRRSV vaccine.
 28. A fusionpolypeptide, comprising: (b) a first translocating peptide fortranslocation; and (c) a first antigenic epitope selected from SEQ IDNOs: 10 and
 12. 29. The fusion polypeptide of claim 28, wherein thefirst translocating peptide is from pseudomonas exotoxin.
 30. The fusionpolypeptide of claim 28, wherein the first translocating peptidecomprises a pseudomonas exotoxin A fragment deleted of only domain III.31. A method for treating or preventing HPV, comprising: administering acomposition to a subject in need, wherein the composition comprises ananti-HPV vaccine and a fusion polypeptide, and the fusion polypeptidecomprises: (b) a first translocating peptide for translocation; and (c)a first antigenic epitope selected from SEQ ID NOs: 10 and
 12. 32. Themethod of claim 31, wherein the first translocating peptide is frompseudomonas exotoxin.
 33. The method of claim 31, wherein the firsttranslocating peptide comprises a pseudomonas exotoxin A fragmentdeleted of only domain III.
 34. The method of claim 31, wherein thecomposition is orally administered to the subject in need.
 35. Themethod of claim 31, wherein the vaccine comprises: a secondtranslocation peptide for translocation and a HPV antigenic epitope. 36.The method of claim 35, wherein the second translocating peptide is frompseudomonas exotoxin.
 37. The method of claim 35, wherein the secondtranslocating peptide comprises a pseudomonas exotoxin A fragmentdeleted of only domain III.